Compositions of toehold primer duplexes and methods of use

ABSTRACT

Provided herein are primers and primer systems having improved specificity and kinetics over existing primers, and methods of use thereof.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/882,231, filed Jul. 1, 2013, which is a national stage filingunder 35 U.S.C. §371 of international application numberPCT/US2011/058178, filed Oct. 27, 2011, which was published under PCTArticle 21(2) in English and claims priority under 35 U.S.C. §119(e)from U.S. provisional application Ser. No. 61/407,291, filed Oct. 27,2010, the entire contents of all of which are incorporated by referenceherein.

FIELD OF INVENTION

The embodiments described herein relate to partially double-strandednucleic acid primers and their use in, for example, nucleic acidsynthesis methods.

BACKGROUND OF INVENTION

Nucleic acids are vital information carriers of biology, and thedetection, amplification, and identification of nucleic acids has formedthe basis for a vast sector of biotechnology. In particular, methodssuch as the polymerase chain reaction (PCR) (Saiki et al. Science 239,487-491 (1988)) have been used all over the world as a reliable means ofamplifying DNA, while reverse transcriptase methods have been used toprobe the transcriptome. The operation of DNA polymerase, RNApolymerase, and reverse transcriptase typically uses a shortoligonucleotide fragment known as a primer to direct the portion of along target to be replicated or transcribed.

Although the specificity of nucleic acid hybridization is frequentlysufficient to direct enzymatic activity for most target sequences,targets with repetitive sequence, secondary structure, and high G/Ccontent are difficult to amplify with high yield. Furthermore, highbackgrounds of other nucleic acids can frequently lead to incorrectamplification, such as in the case of single copy human genomeamplification. Finally, multiplexed amplification, such as from a DNAchip pool, can be difficult to achieve due to the large number oforthogonal amplification reactions that must occur simultaneously.Similar problems exist for transcription and for reverse transcription.

SUMMARY OF INVENTION

The hybridization of nucleic acids is specific at the single nucleotidelevel. For example, cytosine preferentially binds to guanine, andadenine preferentially binds to thymine or uracil. However, for nucleicacid molecules composed of many nucleotides, the specificity ofhybridization is reduced, and nucleic acids with near complementarysequences will bind almost as strongly as perfect complementarysequences. Given a heterogeneous mixture of target nucleic acid ofinterest (“targets”) and nucleic acids with sequences that differ fromthe target by, for example, one nucleotide (“spurious targets”), asignificant portion of primers complementary to the target willhybridize instead to spurious targets.

Because the correct targets bind with a slightly higher affinity to aprimer having a complementary sequence, given enough time, correcttargets will eventually displace spurious targets in binding to acomplementary primer. Though, this process is very slow, and would takemonths at the nanomolar concentrations typical of many experimentalsystems.

In order to mitigate the propensity of complementary primers binding tospurious targets it is often necessary to operate nucleic acidprimer-based experimental systems near the melting temperature of theprimer/target complex. Because this melting temperature is generallymuch higher than the temperature at which most biological systemsnaturally operate, this high temperature requirement precludes theexperimental system from operating under normal biological conditions.Additionally, because the melting temperature will vary from target totarget, the requisite narrow temperature range for such experimentalsystems restricts the simultaneous use of multiple primers to detect aplurality of targets.

Provided herein are primers (e.g., primer duplexes and hairpin primerduplexes) that, in embodiments, are able to rapidly bind to nucleic acidtargets with high specificity at a broad range of temperatures. Theseprimers may be used, for example, in nucleic acid synthesis reactions(e.g., PCR), microarray analyses, imaging methods, and single nucleotidepolymorphism (SNP) analyses. The primers may also be used in nucleicacid detection assays where they function primarily as “probes”.Accordingly, regardless of the application, the primers of the inventionmay be referred herein to interchangeably as “probes”. Regardless of theapplication (or method of use), the primers of the invention overcomeproblems commonly experienced when specific hybridization is required inthe presence of spurious targets, and more particularly when suchspurious targets are present in excess.

The primers provided herein possess several unique properties thatfacilitate their use in combination with enzymes that act upon nucleicacids. First, the primers are thermodynamically designed to bind withhigh specificity to only their intended targets, and they show highdiscrimination against even single-nucleotide changes. Second, thespecificity of the primers enables PCR, transcription, and reversetranscription of traditionally difficult targets, such as those havingsignificant sequence repetition, secondary structure, and/or high G/Ccontent. The high degree of specificity that can be achieved with theseprimers further enables accurate processing even in high nucleic acidbackgrounds such as single-copy human genome amplification. Third, thepartially double-stranded nature of the primers means that they areunlikely to interact with each other, and consequently they are amenableto highly multiplexed replication, transcription, and/or reversetranscription reactions. Finally, the hybridization of these primers totargets is relatively robust to temperature and salinity, and thereforethe primers may be of significantly greater length than standardprimers, which in turn provides further enhanced specificity and primerdesign flexibility.

In some embodiments, the nucleic acid primers discussed here arerationally designed so that the standard free energy for hybridization(e.g., theoretical standard free energy) between the specific targetnucleic acid molecule and the primer is close to zero, while thestandard free energy for hybridization between a spurious target (evenone differing from the specific (actual) target by as little as a singlenucleotide) and the primer is high enough to make their bindingunfavorable by comparison. The inventors accomplished this by designinga primer having (a) a “toehold” single-stranded target specific region,(b) a “branch migration” double-stranded target specific region, and (c)a “balance” double-stranded target non-specific region.

In some embodiments, the primer may be comprised of a single strand thatself hybridizes to form double-stranded regions. In some embodiments,the primer may be comprised of two strands. As an example of the latterembodiment, the primers is comprised of a first or complement strand anda second or protector strand. The complement strand, as its nameimplies, is partially complementary to the target of interest and willhybridize to the target. The protector strand, on the other hand, isdesigned to not hybridize to the target and rather to compete with thetarget (or spurious target) for binding of the complement.

The “toehold” region is present in the complement strand, iscomplementary to a target sequence and not complementary to a protectorregion. The “balance” region in the complement strand (i.e., thecomplement balance region) is complementary to part of the protector(i.e., to the protector balance region) and not complementary to targetsequence. The hybridization energy of toehold to target is matched ornearly matched to the hybridization energy of complement balance regionto protector balance region (adjusting for various other thermodynamicconsiderations). The sequence of the balance region is rationallydesigned to achieve this matching under desired conditions oftemperature and primer concentration. As a result, the equilibrium forthe actual target and primer rapidly approaches 50%target:primer:protector:primer (or whatever ratio is desired), whileequilibrium for the spurious target and primer greatly favorsprotector:primer. The abundant free primer in the presence of specifictarget facilitates its highly sensitive and specific detection.

In some embodiments, the nucleic acid primers discussed here aredesigned so that the concentration-adjusted free energy forhybridization between the specific target nucleic acid molecule and theprimer is close to zero, while the concentration-adjusted standard freeenergy for hybridization between a spurious target and the primer ishigh enough to make their binding unfavorable by comparison.“Concentration-adjusted free energy,” as used herein, refers toΔG°+(Δn)RT ln(c), where R is the universal gas constant, T istemperature in Kelvins, c is concentration of the primer, and Δn is thechange in the number of molecules through the course of the reaction(Δn=−1 for standard hybridization, Δn=0 for two-stranded primerhybridization.

Aspects of the invention therefore provide the primer compositionscomprising the primers, compositions comprising the complement andprotector strands (for example in kits), methods of making the primers,and methods of using the primers in assays or reactions includingwithout limitation nucleic acid synthesis and/or detection assays orreactions.

Thus, in one aspect, the invention provides a partially double-strandedprimer comprised of (a) first nucleic acid strand (also referred toherein as a complement strand) and second nucleic acid strand (alsoreferred to herein as a protector strand), wherein the first and secondstrands when hybridized to each other are arranged into (1) adouble-stranded target-non-specific (balance) region, (2) adouble-stranded target-specific (branch migration) region, and (3) asingle-stranded target-specific (toehold) region contributed to by thefirst nucleic acid strand, wherein the double-strandedtarget-non-specific region has a standard free energy approximatelyequal to the standard free energy for the single-strandedtarget-specific region bound to a target nucleic acid. The partiallydouble-stranded primer may comprise one or more double-strandedtarget-non-specific regions, one or more double-stranded target-specificregions, and/or one or more single-stranded target-specific regions. Insome embodiments, the partially double-stranded primer may comprise oneor two double-stranded target-non-specific (balance) regions, one ormore double-stranded target-specific (branch migration) regions, and/orone or more single-stranded target-specific (toehold) regions.

In some embodiments, the second nucleic acid strand comprises anon-extendable nucleotide at its 3′ end and/or the first nucleic acidstrand comprises a non-natural nucleotide at or near the 3′ end of itstarget-non-specific region. In some embodiments, the non-extendablenucleotide is a non-natural nucleotide or a dideoxy nucleotide. In someembodiments, the non-natural nucleotide is iso-C, iso-G or deoxyuridine.These examples are intended as non-limiting.

In some embodiments, the double-stranded target non-specific region isabout 4-20 nucleotides in length. The double-stranded targetnon-specific region may be longer than 20 nucleotides, such as forexample 4-21 nucleotides in length. In some embodiments, it may be about12-192 nucleotides in length.

In some embodiments, the single stranded target specific region is about4-20 nucleotides in length. The single stranded target specific regionmay be longer than 20 nucleotides, such as for example 4-21 nucleotidesin length. In some embodiments, it may be about 12-192 nucleotides inlength.

In some embodiments, the double-stranded target non-specific region andthe single stranded target specific region have similar or identicalproportions of A/T nucleotides (and typically similar or identicalproportions of G/C nucleotides). In some embodiments, the first andsecond nucleic acid strands are comprised of DNA or RNA or a combinationthereof.

In another aspect, the invention provides a single-stranded primer thatpartially self-hybridizes to form (1) a double-strandedtarget-non-specific region, (2) a double-stranded target-specificregion, (3) single-stranded target-specific region, and (4) a hairpinloop region, wherein the one or more double-stranded target-non-specificregion has a concentration-adjusted standard free energy approximatelyequal to the concentration-adjusted standard free energy for the one ormore single-stranded target-specific region bound to a target nucleicacid.

In another aspect, the invention provides a composition comprising theany of the afore-mentioned primers. The composition may further comprisea carrier such as a buffer, optionally comprising a preservative, one ormore salts, etc. The composition may also comprise an excess ofsingle-stranded protector strands, wherein each protector strandcomprises a protector balance region and a protector branch migrationregion. The single stranded protector strands may each comprise anon-extendable and/or non-naturally occurring nucleotide, preferably atits 3′ end.

In another aspect, the invention provides a system comprising a nucleicacid target, a polymerase, and any of the foregoing primers. In someembodiments, the primer is a partially double-stranded primer comprisinga first and a second nucleic acid strand arranged into (1) adouble-stranded target-non-specific region, (2) a double-strandedtarget-specific region, and (3) a single-stranded target-specific regioncontributed to by the first nucleic acid strand.

In some embodiments, the nucleic acid target is a single-stranded. Insome embodiments, the nucleic acid target is DNA or RNA. In someembodiments, the nucleic acid target comprises repetitive sequence,secondary structure and/or high GC content. In some embodiments, thenucleic acid target is present in a plurality of different nucleicacids. In some embodiments, the nucleic acid target is present as asingle copy or in low copy (e.g., less than 0.001%, less than 0.01%,less than 0.1%, or less than 1%) in a plurality of different nucleicacids.

In some embodiments, the system comprises a plurality of any of theforegoing primers such as a plurality of different partiallydouble-stranded primers. In some embodiments, the system comprises atleast two of the foregoing primers, such as at least two partiallydouble-stranded primers, which together can be used to amplify a regionof the nucleic acid target.

In another aspect, the invention provides a composition comprising theany of the afore-mentioned systems. The composition may further comprisea carrier such as a buffer, optionally comprising a preservative, one ormore salts, one or more enzymes such as a polymerase, nucleotidessuitable for nucleic acid synthesis, etc. The composition may alsocomprise an excess of single-stranded protector strands, wherein eachprotector strand comprises a protector balance region and a protectorbranch migration region. The single stranded protector strands may eachcomprise a non-extendable and/or non-naturally occurring nucleotide,preferably at its 3′ end.

In another aspect, the invention provides a method comprising contactingany of the foregoing primers, including any of the foregoing partiallydouble-stranded primers to a sample, and detecting hybridization of theprimer to a target in the sample.

In some embodiments, the primer such as the partially double-strandedprimer is labeled with a detectable moiety. In some embodiments, thedetectable moiety comprises a fluorophore or a radioisotope.

The target will typically be a nucleic acid. In some embodiments, thetarget is a single-stranded nucleic acid. In some embodiments, thetarget is DNA or RNA. In some embodiments, the target is a nucleic acidthat comprises repetitive sequence, secondary structure and/or high GCcontent. In some embodiments, the target is present in a plurality ofdifferent nucleic acids. In some embodiments, the target is present as asingle copy or in low copy (e.g., less than 0.001%, less than 0.01%,less than 0.1%, or less than 1%) in a plurality of different nucleicacids.

In some embodiments, this and other methods described herein areperformed at a temperature below the melting temperature of thecomplement strand-target complex. In some embodiments, this and othermethods described herein are performed at a temperature between andincluding room temperature up to and including 50° C., or up to andincluding 40° C., or up to and including 30° C. In some embodiments,this and other methods described herein are performed at about 37° C. Insome embodiments, this and other methods described herein are performedin an excess of protector strand that comprises a protector balanceregion and a protector branch migration region and that is identical tothe protector strand in the partially double-stranded primer. In thisand other methods described herein, the primer may be any of theforegoing primers including the partially double-stranded primers.

In another aspect, the invention provides a method comprisinghybridizing a single-stranded target-specific (toehold) region of afirst (complement) strand of any of the foregoing partiallydouble-stranded primers to a nucleic acid target, thereby dissociatingthe first strand of the primer from the second (protector) strand of theprimer, and extending the first strand at its 3′ end, in atarget-complementary manner, in the presence of a polymerase.

In another aspect, the invention provides a method comprising performinga nucleic acid synthesis reaction in the presence of a nucleic acidtarget, a polymerase, and one or more of the foregoing partiallydouble-stranded primers.

In some embodiments, the nucleic acid synthesis reaction is a nucleicacid amplification reaction. In some embodiments, the nucleic acidamplification reaction is polymerase chain reaction (PCR). In someembodiments, the nucleic acid synthesis reaction is a transcriptionreaction. In some embodiments, the transcription reaction is a reversetranscription reaction.

In some embodiments, two partially double-stranded primers are used.

In another aspect, the invention provides a method of performing amultiplexed nucleic acid amplification reaction comprising amplifyingmultiple unique nucleic acid molecules using any of the foregoingprimers including the partially double-stranded primer.

In another aspect, the invention provides a kit comprising one or more(including a plurality) of any of the foregoing partiallydouble-stranded primers, and one or more nucleic acid synthesis reagentssuch as enzymes, nucleotides, salts, EDTA, a buffer, etc.

In some embodiments, the one or more nucleic acid synthesis reagents isselected from the group consisting of a buffer, nucleotides, and apolymerase.

In some embodiments, the kit further comprises an excess of protectorstrand that is identical to the protector strand comprised in theprimer.

In some embodiments, the kit further comprises instructions for use.

In another aspect, the invention provides a kit comprising a firstsingle-stranded (complement) nucleic acid in a first container, and asecond single-stranded (protector) nucleic acid that is complementary toa region of the first single-stranded nucleic acid, in a secondcontainer, wherein, when the first and second single-stranded nucleicacids are hybridized to each other, a partially double-stranded nucleicacid is formed that comprises (1) a double-stranded target-non-specificregion, (2) a double-stranded target-specific region, and (3) asingle-stranded target-specific region contributed to by the firstnucleic acid, wherein the first single-stranded nucleic acid comprises anon-natural nucleotide and/or the second single-stranded nucleic acidcomprises a non-extendable nucleotide at its 3′ end.

In some embodiments, the kit further comprises instructions for use. Insome embodiments, the kit further comprises one or more nucleic acidsynthesis reagents such as those recited above. In some embodiments, theone or more nucleic acid synthesis reagents is selected from the groupconsisting of a buffer, nucleotides, and a polymerase.

In some embodiments, the protector strand is provided in the kit in anamount (e.g., a molar amount) that is greater than the amount (e.g., amolar amount) of complement strand in the kit.

In some embodiments of the foregoing aspects and inventions,particularly those relating to two strand primers, the nucleotidesequence of the primer is selected such that: |ΔG₁°−ΔG₂°−ΔG₃°|≦Δ_(R)°,wherein: ΔG₁° is the standard free energy of hybridization of theprotector balance region to the complement balance region; ΔG₂° is thestandard free energy of hybridization of the protector balance region tothe sequence immediately adjacent in the first direction to the targetnucleic acid sequence, if any; ΔG₃° is the standard free energy ofhybridization of the toehold region to the second target nucleic acidsequence; and ΔG_(R)° is 3.5 kcal/mol.

In one aspect, provided herein is a primer duplex system comprising acomplement strand and a protector strand, wherein the protector strandcomprises a nucleic acid having: a protector branch migration regionhaving a first end, a second end, and a sequence that corresponds to afirst target nucleic acid sequence having a first end and a second end,wherein the first end of the protector branch migration region and thefirst end of the first target nucleic acid sequence are either both 5′or else both 3′; and a protector balance region immediately adjacent tothe first end of the protector branch migration region having a sequencethat does not correspond to sequence immediately adjacent to the firstend of the first target nucleic acid sequence, if any; and thecomplement primer comprises a nucleic acid having: a complement branchmigration region having a first end and a second end, and a sequencethat is complementary to the protector branch migration region, whereinthe first end of the complement branch migration region and the firstend of the first target nucleic acid sequence are either both 5′ or elseboth 3′; a toehold region that is: immediately adjacent to the first endof the complement branch migration region; and complementary to a secondtarget nucleic acid sequence that is immediately adjacent to the secondend of the first target nucleic acid sequence; and a complement balanceregion that: is immediately adjacent to the second end of the complementbranch migration region; is complementary to the protector balanceregion; and has a sequence such that: |Δ₁°−ΔG₂°−ΔG₃°|≦ΔG_(R)°, wherein:ΔG₁° is the standard free energy of hybridization of the protectorbalance region to the complement balance region; ΔG₂° is the standardfree energy of hybridization of the protector balance region to thesequence immediately adjacent in the first direction to the targetnucleic acid sequence, if any; ΔG₃° is the standard free energy ofhybridization of the toehold region to the second target nucleic acidsequence; and ΔG_(R)° is 3.5 kcal/mol.

In another aspect, provided herein is a primer duplex system comprisinga nucleic acid having a protector strand, a hairpin region and acomplement strand, wherein: the protector strand comprises a protectorbranch migration region and a protector balance region, wherein: theprotector branch migration region has: a first end; a second end; and asequence that corresponds to a first target nucleic acid sequence havinga first end and a second end, wherein the first end of the protectorbranch migration region and the first end of the first target nucleicacid sequence are either both 5′ or else both 3′; and the protectorbalance region has: a first end; a second end immediately adjacent tothe first end of the protector branch migration region; and a sequencethat does not correspond to sequence immediately adjacent to the firstend of the first target nucleic acid sequence, if any; the hairpinregion comprises: a first end; and a second end immediately adjacent tothe first end of the protector balance region; and the complement strandcomprises a complement balance region, a complement branch migrationregion, and a toehold region, wherein: the complement balance regionhas: a first end; a second end immediately adjacent to the first end ofthe hairpin region; and a sequence that is complementary to theprotector balance region; the complement branch migration region has: afirst end; a second end immediately adjacent to the first end of thecomplement balance region; and a sequence that is complementary to theprotector branch migration region, wherein the first end of thecomplement branch migration region and the first end of the first targetnucleic acid sequence are either both 5′ or else both 3′; the toeholdregion is: immediately adjacent to the first end of the complementbranch migration region; and complementary to a second target nucleicacid sequence that is immediately adjacent to the second end of thefirst target nucleic acid sequence; and the complement balance regionhas a sequence such

that: |ΔG₁−ΔG₂°−ΔG₃°+ΔG₄°+RT ln(c)|≦ΔG_(R)°, wherein: ΔG₁° is thestandard free energy of hybridization of the protector balance region tothe complement balance region; ΔG₂° is the standard free energy ofhybridization of the protector balance region to the sequenceimmediately adjacent in the first direction to the target nucleic acidsequence, if any; and ΔG₃°; is the standard free energy of hybridizationof the toehold region to the second target nucleic acid sequence; ΔG₄°is the standard free energy of confinement of the hairpin region; R isthe ideal gas constant; T is the temperature at which the primer duplexsystem is to be used; c is the concentration at which the primer duplexsystem is to be used; and ΔG_(R)° is 3.5 kcal/mol.

In yet another aspect, provided herein system having, in 3′ to 5′ order,a first protector strand, a first hairpin region, a complement strand, asecond hairpin region and a second protector strand, wherein: the firstprotector strand comprises: a first protector branch migration regionhaving a sequence that corresponds to a first target nucleic acidsequence; and a first protector balance region that: is immediately 5′to the first protector branch migration region; and has a sequence thatdoes not correspond to sequence immediately 5′ to the first targetnucleic acid sequence, if any; the first hairpin region is immediately5′ to the first protector balance region; the complement strandcomprises: a first complement balance region that: is immediately 5′ tothe first hairpin region; and has a sequence complementary to thesequence of the first protector balance region; a first complementbranch migration region that: is immediately 5′ to the first complementbalance region; and has a sequence complementary to a first protectorbranch migration region; a toehold region that: is immediately 5′ to thefirst complement branch migration region; and has a sequence that iscomplementary to a second target nucleic acid sequence that isimmediately 3′ to the first target nucleic acid sequence; a secondcomplement branch migration region that: is immediately 5′ to thetoehold region; and has a sequence complementary to a third targetnucleic acid sequence that is immediately 3′ to the second targetnucleic acid sequence; a second complement balance region that: isimmediately 5′ to the second complement branch migration region; has asequence that is not complementary to sequence immediately 3′ to thethird target nucleic acid sequence, if any; the second hairpin region isimmediately 5′ to the second complement balance region; and the secondprotector strand comprises: a second protector balance region that: isimmediately 5′ to the second hairpin region; and has a sequencecomplementary to the second complement balance region; and a secondprotector branch migration region that: is immediately 5′ to the secondprotector balance region; and has a sequence complementary to the secondcomplement branch migration region; wherein the first complement balanceregion and the second complement balance region have sequences suchthat: |ΔG₁°−ΔG₂°+ΔG₃°−ΔG₄°−ΔG₅°+ΔG₆°+RT ln(c)|≦ΔG_(R)°, wherein: ΔG₁+ isthe standard free energy of hybridization of the first protector balanceregion to the first complement balance region; ΔG₂° is the standard freeenergy of hybridization of the first complement balance region to thesequence immediately 5′ to the first target nucleic acid sequence, ifany; ΔG₃° is the standard free energy of hybridization of the secondprotector balance region to the second complement balance region; ΔG₄°is the standard free energy of hybridization of the second complementbalance region to the sequence immediately 3′ to the third targetnucleic acid sequence, if any; ΔG₅° is the standard free energy ofhybridization of the toehold region to the second target nucleic acidsequence; ΔG₆° is the standard free energy of confinement of the firsthairpin region; ΔG₇° is the standard free energy of confinement of thesecond hairpin region; R is the ideal gas constant; T is the temperatureat which the primer duplex system is to be used; and c is theconcentration at which the primer duplex system is to be used; andΔG_(R)° is 3.5 kcal/mol.

In still another aspect, provided herein is a primer duplex systemcomprising a hairpin primer and a protector strand, wherein: the hairpinprimer comprises a nucleic acid having: a first protector strand having:a first protector branch migration region having a sequence thatcorresponds to a first target nucleic acid sequence; and a firstprotector balance region that: is immediately 5′ to the first protectorbranch migration region; and has a sequence that does not correspond tosequence immediately 5′ to the first target nucleic acid sequence, ifany; a hairpin region immediately 5′ to the first protector balanceregion; a complement strand having: a first complement balance regionthat: is immediately 5′ to the first hairpin region; and has a sequencecomplementary to the sequence of the first protector balance region; afirst complement branch migration region that: is immediately 5′ to thefirst complement balance region; and has a sequence complementary to afirst protector branch migration region; a toehold region that: isimmediately 5′ to the first complement branch migration region; and hasa sequence that is complementary to a second target nucleic acidsequence that is immediately 3′ to the first target nucleic acidsequence; a second complement branch migration region that: isimmediately 5′ to the toehold region; and has a sequence complementaryto a third target nucleic acid sequence that is immediately 3′ to thesecond target nucleic acid sequence; a second complement balance regionthat: is immediately 5′ to the second complement branch migrationregion; has a sequence that is not complementary to sequence immediately3′ to the third target nucleic acid sequence, if any; and the protectorcomprises a nucleic acid having: a second protector strand having: asecond protector balance region that has a sequence complementary to thesecond complement balance region; and a second protector branchmigration region that: is immediately 5′ to the second protector balanceregion; and has a sequence complementary to the second complement branchmigration region; wherein the first complement balance region and thesecond complement balance region have sequences such that:|ΔG₁°−ΔG₂°+ΔG₃°−ΔG₄°−ΔG₅°+ΔG₆°|≦ΔG_(R)°, wherein: ΔG₁° is the standardfree energy of hybridization of the first protector balance region tothe first complement balance region; ΔG₂° is the standard free energy ofhybridization of the first complement balance region to the sequenceimmediately 5′ to the first target nucleic acid sequence, if any; ΔG₃°is the standard free energy of hybridization of the second protectorbalance region to the second complement balance region; ΔG₄° is thestandard free energy of hybridization of the second complement balanceregion to the sequence immediately 3′ to the third target nucleic acidsequence, if any; ΔG₅° is the standard free energy of hybridization ofthe toehold region to the second target nucleic acid sequence; ΔG₆° isthe standard free energy of confinement of the hairpin region; andΔG_(R)° is 3.5 kcal/mol.

In a further aspect, provided herein is a primer duplex systemcomprising a protector strand and a hairpin primer, wherein: theprotector strand comprises a nucleic acid having: a first protectorstrand having: a first protector branch migration region having asequence that corresponds to a first target nucleic acid sequence; and afirst protector balance region that: is immediately 5′ to the firstprotector branch migration region; and has a sequence that does notcorrespond to sequence immediately 5′ to the first target nucleic acidsequence, if any; the hairpin primer comprises a nucleic acid having: acomplement strand having: a first complement balance region that has asequence complementary to the sequence of the first protector balanceregion; a first complement branch migration region that: is immediately5′ to the first complement balance region; and has a sequencecomplementary to a first protector branch migration region; a toeholdregion that: is immediately 5′ to the first complement branch migrationregion; and has a sequence that is complementary to a second targetnucleic acid sequence that is immediately 3′ to the first target nucleicacid sequence; a second complement branch migration region that: isimmediately 5′ to the toehold region; and has a sequence complementaryto a third target nucleic acid sequence that is immediately 3′ to thesecond target nucleic acid sequence; a second complement balance regionthat: is immediately 5′ to the second complement branch migrationregion; has a sequence that is not complementary to sequence immediately3′ to the third target nucleic acid sequence, if any; a hairpin regionimmediately 5′ to the second complement balance region; and a secondprotector strand having: a second protector balance region that: isimmediately 5′ to the second hairpin region; and has a sequencecomplementary to the second complement balance region; and a secondprotector branch migration region that: is immediately 5′ to the secondprotector balance region; and has a sequence complementary to the secondcomplement branch migration region; wherein the first complement balanceregion and the second complement balance region have sequences suchthat: |ΔG₁°−ΔG₂°+ΔG₃°−−ΔG₄°−ΔG₅°+ΔG₆°|≦ΔG_(R)°, wherein: ΔG₁° is thestandard free energy of hybridization of the first protector balanceregion to the first complement balance region; ΔG₂° is the standard freeenergy of hybridization of the first complement balance region to thesequence immediately 5′ to the first target nucleic acid sequence, ifany; ΔG₃° is the standard free energy of hybridization of the secondprotector balance region to the second complement balance region; ΔG₄°is the standard free energy of hybridization of the second complementbalance region to the sequence immediately 3′ to the third targetnucleic acid sequence, if any; and ΔG₅° is the standard free energy ofhybridization of the toehold region to the second target nucleic acidsequence; ΔG₆° is the standard free energy of confinement of the hairpinregion; and ΔG_(R)° is 3.5 kcal/mol.

In another aspect, provided herein is a primer duplex system comprisinga first protector strand, a complement strand and a second protectorstrand, wherein: the first protector strand comprises a nucleic acidhaving: a first protector branch migration region having a sequence thatcorresponds to a first target nucleic acid sequence; and a firstprotector balance region that: is immediately 5′ to the first protectorbranch migration region; and has a sequence that does not correspond tosequence immediately 5′ to the first target nucleic acid sequence, ifany; the complement strand comprises a nucleic acid having: a firstcomplement balance region that: is immediately 5′ to the first hairpinregion; and has a sequence complementary to the sequence of the firstprotector balance region; a first complement branch migration regionthat: is immediately 5′ to the first complement balance region; and hasa sequence complementary to a first protector branch migration region; atoehold region that: is immediately 5′ to the first complement branchmigration region; and has a sequence that is complementary to a secondtarget nucleic acid sequence that is immediately 3′ to the first targetnucleic acid sequence; a second complement branch migration region that:is immediately 5′ to the toehold region; and has a sequencecomplementary to a third target nucleic acid sequence that isimmediately 3′ to the second target nucleic acid sequence; a secondcomplement balance region that: is immediately 5′ to the secondcomplement branch migration region; has a sequence that is notcomplementary to sequence immediately 3′ to the third target nucleicacid sequence, if any; and the second protector strand comprises: asecond protector balance region that has a sequence complementary to thesecond complement balance region; and a second protector branchmigration region that: is immediately 5′ to the second protector balanceregion; and has a sequence complementary to the second complement branchmigration region; wherein the first complement balance region and thesecond complement balance region have sequences such that:|ΔG₁°−ΔG₂°+ΔG₃°−ΔG₄°−ΔG₅°−RT ln(c)|≦ΔG_(R)°, wherein: ΔG₁° is thestandard free energy of hybridization of the first protector balanceregion to the first complement balance region; ΔG₂° is the standard freeenergy of hybridization of the first complement balance region to thesequence immediately 5′ to the first target nucleic acid sequence, ifany; ΔG₃° is the standard free energy of hybridization of the secondprotector balance region to the second complement balance region; ΔG₄°is the standard free energy of hybridization of the second complementbalance region to the sequence immediately 3′ to the third targetnucleic acid sequence, if any; and ΔG₅° is the standard free energy ofhybridization of the toehold region to the second target nucleic acidsequence; R is the ideal gas constant; T is the temperature at which theprimer duplex system is to be used; c is the concentration at which theprimer duplex system is to be used; and ΔG_(R)° is 3.5 kcal/mol.

In yet another aspect, provided herein is a primer duplex systemcomprising, in 3′ to 5′ order, a first protector strand, a first hairpinregion, a complement strand, a second hairpin region and a secondprotector strand, wherein: the first protector strand has a sequencethat corresponds to a first target nucleic acid sequence; the firsthairpin region is immediately 5′ of the first protector strand; thecomplement strand comprises: a first complement branch migration regionthat: is immediately 5′ of the first hairpin region; and has a sequencecomplementary to the sequence of the first protector strand; a toeholdregion that: is immediately 5′ of the first complement branch migrationregion; and has a sequence complementary to a second target nucleic acidsequence that is immediately 3′ of the first target nucleic acidsequence; and a second complement branch migration region that: isimmediately 5′ of the toehold region; and has a sequence complementaryto a third target nucleic acid sequence that is immediately 3′ of thesecond nucleic acid sequence; the second hairpin region is immediately5′ of the second complement branch migration region; and the secondprotector strand has a sequence that is complementary to the sequence ofthe second complement branch migration region.

In any one of the foregoing aspects, ΔG_(R)° may be 2.0 kcal/mol, 1.0kcal/mol, or 0.5 kcal/mol; and/or c may be about 10 nM; and/or T may beabout 293 K or about 338 K; and/or the toehold region may be between 4and 20 nucleotides in length, between about 4 and 15 nucleotides inlength, or between about 4 and 10 nucleotides in length; and/or thefirst end of the protector branch migration region may be 5′ or 3′;and/or the primer duplex system may further comprise a functionalizedfluorescent group or dye; and/or the primer duplex system may beimmobilized on a solid support; and/or the hairpin region may be nogreater than 20 nucleotides in length or no greater than 10 nucleotidesin length; and/or the sequence of the hairpin region may be selectedfrom the group consisting of a poly-adenosine sequence,poly-deoxyadenosine sequence, a poly-5′-methyluridine sequence, apoly-thymidine sequence, a poly-guanosine sequence, apoly-deoxyguanosine sequence, a poly-cytidine sequence, apoly-deoxycytidine sequence, a poly-uridine sequence, and apoly-deoxyuridine sequence; and/or the first target nucleic acidsequence and/or the second target nucleic acid sequence may be sequencesthat naturally occur in an organism or a virus; and/or the first targetnucleic acid sequence and/or the second target nucleic acid sequence maybe sequences that naturally occur in a micro-RNA.

In one aspect, provided herein is a method of detecting a target nucleicacid in a sample comprising: contacting a target nucleic acid with aprimer duplex system of any one of the embodiments described herein; anddetecting the formation of a complex between the target nucleic acid andat least a part of the primer duplex system. In some embodiments, theprimer duplex system further comprises a functionalized fluorescentgroup or dye. In some embodiments, the primer duplex system isimmobilized on a solid support. In some embodiments, the contactingoccurs in a cell. In some embodiments, the target nucleic acid is anucleic acid that naturally occurs in an organism or a virus. In someembodiments, the target nucleic acid is a micro-RNA.

In another aspect, provided herein is a method of amplifying a sequencecontained within a target nucleic acid comprising: forming a solutioncomprising: a target nucleic acid; a primer duplex system of any one ofthe embodiments described herein; and reagents for performing anamplification reaction; and incubating the solution under conditionssuch that a sequence contained within the target nucleic acid isamplified. In some embodiments, the target nucleic acid is a nucleicacid that naturally occurs in an organism or a virus.

These and other aspects and embodiments of the invention will beexplained in greater detail herein.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A, 1B, 2A, 2B, 3A, 3B, 4-8, 9A, and 9B depict exemplary nucleicacid probe systems.

FIGS. 10, 11, 12A, 12B, 13, and 14 depict exemplary methods of usingnucleic acid probe systems.

FIGS. 15A and 15B depict highly specific polymerase chain reaction (PCR)using the primer duplexes provided herein.

FIGS. 16A-16E show experimental demonstrations of primer hybridizationwith single nucleotide discrimination.

FIGS. 17A-17D show additional experimental results and statistics on thesingle-base discrimination abilities of primer duplexes.

FIGS. 18A-18B show experimental results using duplex primers to improvethe PCR yield of a quasi-repetitive target.

DETAILED DESCRIPTION OF INVENTION

A significant challenge in probe-based nucleic acid assays is thatnucleic acids having sequences similar to that of a target willhybridize to the target's complement with strong thermodynamics and fastkinetics. However, as described herein, the kinetics and thermodynamicsof strand displacement reactions can be partially decoupled, so thatreactions that are only slightly thermodynamically favorable or evenunfavorable can nonetheless have kinetics as fast as the hybridizationof two complementary strands. The compositions and methods describedherein take advantage of this decoupling mechanism to provide nucleicacid probe systems with improved specificity and kinetics.

Provided herein are highly specific nucleic acid probe systems andmethods of using such probe systems. In certain embodiments, the nucleicacid probe systems described herein comprise complement probes havingregions complementary to a target sequence that are protected fromhybridization to spurious targets by protector regions complementary toa portion of the complement probes. The free energy of the bindingreaction between the target and the protected probe is finely controlledvia the rationally designed bases of one or more balancing regions,which have sequences that do not correspond to the target nucleic acidsequence or its complement. In certain embodiments, a protector and acomplement probe form regions on a single nucleic acid molecule and areseparated from one another by one or more nucleic acid hairpins.

The methods and compositions described herein possess several uniqueproperties that facilitate their use in hybridization assays. First, thenucleic acid probe systems described herein reliably convert smallsequence differences between targets and spurious targets into largedifferences in binding affinity and reaction rates between hybridizationof the target vs. spurious target with the probe. Second, the nucleicacid probe systems described herein can be designed to operate at any ofa wide range of temperatures and salt concentrations, and can thereforefunction reliably under many different experimental conditions. Third,use of the nucleic acid probe systems described herein can result inhybridization reactions that are kinetically fast even at roomtemperature, which facilitates rapid and high-throughput analysis ofnucleic acids. Fourth, the nucleic acid probe systems described hereinare rationally designed, and therefore are unlikely to interactunfavorably or in unexpected ways with other biomolecules.

Accordingly, provided herein are primer compositions, methods of makingsuch compositions, and methods of their use. The embodiments describedherein are premised in part on the discovery that primer (e.g., a pairof partially hybridized primers, or a single self-hybridizing primer)that are partially double-stranded and partially single-stranded, whenused in a nucleic acid synthesis reaction for example, are able todiscriminate between fully complementary targets and those having one ormore mismatches (i.e., spurious targets). As demonstrated herein, theprimer duplexes described herein are superior to standard primers in,for example, PCR reactions using spurious targets such as those havingquasi-repetitive sequences.

The primer duplexes herein comprise a single-stranded region referred toherein as a “toehold” from which the primer duplex initiates binding toa target, a double-stranded “balance region” which spontaneouslydissociates so that a single primer strand does not completehybridization (along the full length of the primer) to the target, and adouble-stranded branch migration region, in between the toehold andbalance regions, which is fully complementary to a target nucleic acidsequence. Mechanistically, it is thought that hybridization to a targetbegins at the toehold and continues along the length of the complementstrand until the primer is no longer “double-stranded”. This assumescomplementarity between the target and the branch region as well. Asused herein, a nucleic acid “region” or “domain” is a consecutivestretch of nucleotides of any length. When nucleotide mismatches existbetween the “target” and the complement strand, displacement of thesecond strand (i.e., the protector strand) is thermodynamicallyunfavorable and the association between the complement strand and the“target” is reversed. It is to be understood that in this latterdescription, the “target” is actually a spurious target since itcomprises nucleotide differences or mismatches from the complementstrand.

Because the standard free energy favors a complete match (fullycomplementary) between the target sequence of the nucleic acid andbranch migration plus toehold regions of the primer rather than amismatch (e.g., single nucleotide change), the first (complement) strandof the primer will bind stably to a target in the absence of a mismatchbut not in the presence of a mismatch. If a mismatch exists between thefirst (complement) strand of the primer and the target, the primerduplex prefers to reform via newly exposed single-stranded balanceregions. In this way, the frequency of beginning a nucleic acidsynthesis reaction at an incorrect position in a target (or in a sample,for that matter) is reduced. This type of discrimination is typicallynot possible using the standard single-stranded primers of the prior artbecause in those reactions there is no competing nucleic acid strand(such as the protector strand) to which a mismatched primer strand wouldprefer to bind. In some embodiments, the primers described herein may besignificantly longer than conventional primers (e.g., those used forpolymerase chain reaction (PCR) amplification) because the instantprimers rely on the presence of a competing, protector strand forspecificity rather than on melting temperature to discriminate betweencomplementary and mismatched sequences. Accordingly, the instant primersmay be selected and used in a manner that is temperature independent.

The primer duplexes described herein therefore improve specificity offor example nucleic acid synthesis reactions and, in some embodiments,allow for a greater degree of multiplexing of primers. Preliminaryexperiments, the results of which are provided herein, show that the PCRyield of quasi-repetitive targets can be significantly improved usingthe primer duplexes provided herein as compared to standard primers(e.g., 75% vs. 30%). The primer duplexes described herein also providefor specific nucleic acid detection and amplification from aheterogeneous population of nucleic acids, such as for example,detecting and amplifying a bacterial DNA from a sample comprised ofhuman DNA, which has broad applicability in detection of rare organismssuch as biowarfare agents.

Primer Duplexes

As used herein, the primers of the invention may be referred to as“primer duplexes” to covey that they may be provided and/or exist in aconformation in which they comprise double-stranded regions.Accordingly, the terms “primer” and “primer duplex” may be usedinterchangeably.

The primer duplexes provide improved specificity and kinetics overexisting primers. A “primer duplex” herein refers to a primer comprisinga first strand (referred to herein as a “complement strand”) and asecond strand (referred to herein as a “protector strand”) partiallycomplementary to the first strand. In some embodiments, the complementstrand and the protector strand are separate single-stranded nucleicacid molecules (FIGS. 1A and 1B). In other embodiments, the complementstrand and the protector strand are connected to each other andseparated by a hairpin region to form contiguous regions of a singlenucleic acid molecule (FIGS. 2A and 2B). As used herein, a “hairpinregion” is a single-stranded loop of nucleotides connecting twodouble-stranded regions of a nucleic acid. The general structure ofexemplary primer duplexes is illustrated in the Figures and describedherein. It is to be understood that, in most instances, when referenceis made to a complement region or a protector region (or vice versa),each region is typically within a single “primer duplex” (or a singleprimer system). For example, a complement balance region in a primer ofthe invention is complementary to a protector balance region in the sameprimer such that a complement balance region of one primer of theinvention does not hybridize to a protector balance region of differentphysically separate primer.

In embodiments in which the primer of the invention consists of only asingle strand, the complement “strand” may be referred to as thecomplement region, and the protector “strand” may be referred to as theprotector region.

In certain embodiments, the complement strand (or region) comprises atoehold region, a complement branch migration region, and a complementbalance region, while the protector strand (or region) comprises aprotector branch migration region and a protector balance region. Asused herein, a nucleic acid “region” is a consecutive stretch ofnucleotides of any length. Toehold and branch migration regions are eachdesigned to be complementary to, and thus “base-pair” with (e.g.,hybridize to), adjacent regions in a target nucleic acid. A region of acomplement strand that base-pairs with a region in a target nucleic acidis referred to as a “target-specific” region. Balance regions aredesigned to be not complementary to, and thus to not base-pair with, atarget nucleic acid. Balance regions therefore are referred to as“target-non-specific” regions. In certain aspects, when the complementstrand (or region) and the protector strand (or region) are hybridizedto each other (are double-stranded), a primer duplex is formed. Thus, insome aspects, a primer duplex comprises a target-specificsingle-stranded toehold region, a target-specific double-stranded branchmigration region, and a target-non-specific double-stranded balanceregion (FIGS. 1A and 1B). In some instances, the primer duplex may alsocomprise a hairpin loop, as described in greater detail below.

The primer duplexes described herein may be designed to hybridizespecifically with a target nucleic acid. The efficacy of a primer, forexample, in a nucleic acid amplification reaction, depends on thespecificity, efficiency, and fidelity of the primer. Typical nucleicacid primers often bind to spurious targets with a thermodynamic andkinetic profile comparable to that of the same primer binding to itsintended, specific target nucleic acid, except between the meltingtemperatures of the mismatched duplex and the perfectly hybridizedduplex. Accordingly, mismatched and perfect duplexes can bedistinguished by their melting temperatures. The primers of theinvention, in contrast, distinguish between spurious and true target ina relatively temperature-independent manner.

A “spurious target” herein refers to a nucleic acid molecule thatdiffers from a target nucleic acid molecule by at least one nucleotidewithin the region hybridizing to the complement strand. For example,TCGACGGGG is a spurious target, if the target is TCGAAGGGG. In certainembodiments, a spurious target comprises at least 2, at least 3, atleast 4, or more nucleotide changes relative to the target. Primerbinding to spurious targets reduces the fidelity (accuracy) of, e.g.,nucleic acid amplification. The primer duplexes presented herein aredesigned to alter the standard free energy of strand displacement withspurious targets, permitting discrimination between correct targets andspurious targets, including spurious targets that differ from a correcttarget by only one nucleotide. As described herein, the protector strandis responsible for altering the standard free energy to allow thecomplement strand to discriminate between correct and spurious targets.

The primers described herein are rationally designed to facilitatestrand displacement reactions with finely tuned kinetics andthermodynamics such that kinetics and thermodynamics of stranddisplacement reactions are partially decoupled. As a result of thisdecoupling, reactions only slightly thermodynamically favorable or evenunfavorable can nonetheless have kinetics as fast as the hybridizationof two complementary strands.

For example, at 37° C. and 1 M Na⁺, the concentration-adjusted standardfree energy for hybridization of a primer to a perfectly complementary(correct or specific) target (i.e., 100% nucleotide match) is between1.9 kcal/mol and 6.6 kcal/mol more favorable than theconcentration-adjusted standard free energy for hybridization of thesame primer to a spurious target for every nucleotide that the spurioustarget differs from the intended target. In certain embodiments, thepresent primer duplexes use toehold exchange strand displacementreactions to translate this 1.9 to 6.6 kcal/mol difference inconcentration-adjusted standard free energy to an optimal discriminationbetween the target and spurious targets. An example of thethermodynamics/kinetics of primer duplex binding to a target nucleicacid is described as follows in reference to FIGS. 3A and 3B.

For purposes of this example, the target nucleic acid has at least tworegions, (1) and (2). In certain embodiments, region (1) may be about 10to about 200 (including 14-200 or 20-200) nucleotides long, while region2 may be smaller, for example, about 4 to about 20 nucleotides long. Asused herein, the terms “nucleotide” and “bases” are usedinterchangeably. The protector strand includes a protector branchmigration region adjacent to a protector balance region (3). Theprotector branch migration region corresponds to target region 1, whilethe protector balance region (3) does not correspond to region (1) orregion (2) or any region immediately 5′ of the target regions. A nucleicacid sequence, domain or region is “immediately adjacent to”,“immediately 5′” or “immediately 3′” to another sequence if the twosequences are part of the same nucleic acid molecule and if no basesseparate the two sequences. The complement strand includes a complementbalance region (3), a complement branch migration region (1), and atoehold region (2). The complement balance region (3) is complementaryto the protector balance region (3), the complement branch migrationregion (1) is complementary to the protector branch migration region andtarget region (1) (i.e., the protector branch migration region and thetarget region (1) are identical in sequence and this both bind to thecomplement branch migration region (1), and the toehold region (2) iscomplementary to target region (2).

In certain embodiments, the balance region is designed so that itsconcentration-adjusted standard free energy (ΔG_(3:3) °) is the same orabout the same as the concentration-adjusted standard free energy forthe toehold region bound to target region (2) (ΔG_(2:2) °). In someinstances, for a 10 nanomolar (nM) primer used in a reaction at 37° C.,|ΔG_(2:2) °| and |ΔG_(3:3) °| (the vertical bars denoting absolutevalue) should each be less than about 11.3 kcal/mol to ensuredissociation of the full protector strand from the target.

In some embodiments, when the primer duplex interacts with a specific(correct) target nucleic acid molecule (FIG. 3A), the dissociation of(3):(3) and the association of (2):(2) balance one another, and the(1):(1) hybridization thermodynamics are identical for the targetnucleic acid and for the protector strand interacting with thecomplement strand. The total free energy change between the two statesis relatively small (e.g., about 1 kcal/mol), and the reaction quickly(e.g., less than a minute) reaches an equilibrium of about 50:50. Incertain embodiments, the balance region may be designed to have standardfree energy very close to that of the toehold region binding to targetregion 2, so that the equilibrium balance is, for example, 60:40 or70:30. In some embodiments, the design of the balance region may alsotake into account other contributors to free energy change during thereaction, such as hybridization between the protector balance region andupstream target sequences (which in some instances is negligible),confinement of a hairpin (if present), intended temperature of use, andintended primer concentration.

In some embodiments, when the primer duplex instead interacts with aspurious target nucleic acid molecule (FIG. 3B), the dissociation of(3):(3) and the association of (2m):(2) are not balanced becausespurious target region (2m) is not fully complementary to the toeholdregion. The equilibrium is consequently shifted to the state in whichthe primer duplex does not bind the spurious target.

Explained another way, in some instances, the free energy of thecomplement strand bound to the protector strand is ΔG3:3°+ΔG_(1:1) °(ignoring contribution from the optional hairpin region and otherconsiderations), which balances the free energy of the complement strandbound to specific target, ΔG_(2:2) °+ΔG_(1:1) °. In this example, thisis because the balance region (3) of the primer duplex has been designedto have a concentration-adjusted standard free energy equal to (orapproximately equal to) that of target region (2). When the primerduplex interacts with a spurious target having a single-nucleotide(base) change in target region (2m), the system's free energy ΔG_(2m:2)°+ΔG_(1:1) ° is less negative than that of the primer duplex, andtherefore disfavored in equilibrium.

As used herein, the term “approximately equal to” in reference tostandard free energy means that the first referenced free energy iswithin 10% of the second referenced free energy. In some embodiments, afirst free energy that is approximately equal to a second free energy iswithin about +3 kcal/mol to about −3 kcal/mol of the second free energy.It is to be understood that the differences between the first and secondtrue energies may be less than or about 1 kcal/mol, less than or about 2kcal/mol, less than or about 3 kcal/mol, less than or about 3.5kcal/mol, or more, in some embodiments.

Although FIG. 3B illustrates a single nucleotide change corresponding toregion (2)/(2m) of a target nucleic acid molecule, the present primerduplexes can also discriminate between a specific target and a spurioustarget having a nucleotide change in region (1). When the spurioustarget has a single-base change in target region 1, then the primerduplex's standard free energy after binding becomes ΔG_(2:2)°+ΔG_(1m:1)°, where ΔG_(1m:1) ° is the standard free energy of themismatched target region (1) binding to the primer duplex's complementregion (1). Because of the single-base change, the primer duplex's freeenergy is less negative than ΔG_(3:3) °+ΔG_(1:1) ° (free energy ofcomplement primer bound to protector), so equilibrium is shifted to thestate in which the primer duplex does not bind the spurious targetregion. Standard free energies can be calculated theoretically based onthe knowledge in the art and the teachings provided herein.

Complement and Protector Strands, Regions or Domains

The complement domains of the nucleic acid probe systems describedherein each include a plurality of regions, including a toehold regionand one or more complement target regions. Both the toehold region andthe one or more complement target regions have nucleic acid sequencesthat are complementary to nucleic acid sequences of the target nucleicacid. The toehold region and the complement target region are thereforeable to base-pair with and therefore form a complex with a sequence of atarget nucleic acid when the nucleic acid probe system is contacted witha target nucleic acid under appropriate hybridization conditions. Thecomplement domains may also include one or more complement balanceregions. The one or more complement balance regions are rationallydesigned. Thus, the sequences of the one or more complement balanceregions are not designed to be complementary to a target nucleic acidsequence.

A toehold region is complementary to (and thus hybridizes to) a sequencein the target nucleic acid molecule; however, a toehold region does nothybridize to a protector strand. Thus, when the complement strand ishybridized to the target nucleic acid molecule, the toehold region isalso hybridized to the target nucleic acid molecule, but when thecomplement strand is hybridized to the protector strand, the toeholdregion remains single-stranded. A toehold region may be positioned atthe 3′ end or the 5′ end of the complement strand (e.g., is an extensionof the 3′ end or 5′ end of the complement strand).

In certain embodiments, a toehold region is about 4 nucleotides to about20 nucleotides in length, about 4 nucleotides to about 15 nucleotides inlength, or about 4 nucleotides to about 10 nucleotides in length. Insome embodiments, a toehold region is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In someembodiments, the toehold region is greater than 20 nucleotides inlength, including for example less than or about 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides.

The complement branch migration region is complementary to a sequence inthe target nucleic acid molecule and to the protector branch migrationregion. Thus, when the complement strand hybridizes to a target nucleicacid molecule, the complement branch migration region hybridizes to thetarget nucleic acid. When the complement strand hybridizes to itsprotector strand, the complement branch migration region hybridizes tothe protector branch migration region.

In certain embodiments, a branch migration region is no more than 200,100, 75, 50, 40, 30, 25 or 20 nucleotides in length. In someembodiments, a branch migration region is about 10 nucleotides to about200 nucleotides in length. In certain embodiments, a branch migrationregion is about 10 nucleotides to about 150 nucleotides, about 10nucleotides to about 100 nucleotides, or about 10 nucleotides to about50 nucleotides in length. In particular embodiments, a branch migrationregion is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, or 200 nucleotides in length. In particularembodiments, a branch migration region may be more than 200 nucleotidesin length, depending on the target nucleic acid molecule of interest.

The balance regions of a complement strand and a protector strand arecomplementary to each other (i.e., form a double-stranded nucleic acid)but are non-complementary to the target of interest (i.e., neither formsa double-stranded nucleic acid with the target). Thus, when a complementstrand hybridizes to a target nucleic acid molecule, the complementbalance region does not hybridize to the target nucleic acid molecule.When the complement strand hybridizes to its protector strand, thecomplement balance region hybridizes to the protector balance region.

The design of the balance region is dependent on the design of thetoehold region. In some embodiments, the balance region is designed suchthat the thermodynamic profile of the balance region is comparable tothat of the toehold region. In some embodiments, the thermodynamicprofile is based on a theoretic model, using for example, Mfold softwareavailable at the bioinfo website of RPI. The number and/or nature ofnucleotides within a balance region is comparable to that of the toeholdregion. For example, if a toehold region is comprised of 40% A and Tnucleotides and 60% G and C nucleotides, then the balance region shouldalso be comprised of 40% A and T nucleotides and 60% G and Cnucleotides. In embodiments, the balance region is designed such that nomore than three consecutive nucleotides are complementary to a sequenceon the target nucleic acid to avoid binding of the balance region to thetarget nucleic acid.

In some embodiments, the length of a balance region is short enough sothat the complement and protector spontaneously dissociate from eachother. In some embodiments, a balance region is about 4 nucleotides toabout 20 nucleotides in length, about 4 nucleotides to about 15nucleotides in length, or about 4 nucleotides to about 10 nucleotides inlength. In some embodiments, a balance region is 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In someembodiments, a balance region is greater than 20 nucleotides, includingfor example less than about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100 or more nucleotides. In some embodiments, the numberof consecutive nucleotides that are complementary to a nucleotidesequence within the target nucleic acid may be greater than threeprovided that the balance region does not bind to the target nucleicacid.

In some embodiments, for example those where the primer duplex containstwo separate nucleic acid strands, the design of a balance region doesnot depend on the concentration of the primer duplex or the temperatureat which the primer duplex is formed/used. In some embodiments, abalance region is designed such that the standard free energy for thereaction in which the protector strand is displaced from the complementstrand by the target nucleic acid molecule is close to zero kcal/mol. Asused herein, “close to zero” means the standard free energy for thereaction is within 3.5 kcal/mol from 0 kcal/mol. In certain embodiments,the standard free energy of this displacement reaction is within 3.5,3.0, 2.5, 2.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 kcal/molof zero kcal/mol.

In other embodiments, for example those where the primer duplexes isformed by a single nucleic acid molecule (e.g., a hairpin regionseparating the complement strand (or region or domain) and the protectorstrand (or region or domain)), the design of a balance region will bedependent on the primer duplex concentration as well as reactiontemperature. In such embodiments, a balance region is designed so thatthe standard free energy for the reaction in which the protector strandis displaced from the complement strand by the target nucleic acid plusRT ln(c) is close to zero kcal/mol, where R is the universal gasconstant (0.0019858775(34) kcal/mol·K), T is the temperature at whichthe primer duplex is used, and c is the concentration at which primerduplex is used. In some embodiments, the temperature at which the primerduplexes are used is about 273 K (0° C.), 277 K, 283 K, 288 K, 293K, 298K, 303 K, 308 K, 313 K, 318 K, 323 K, 328 K, 333 K, 338 K, 343 K, 348 K,353 K, 358 K or 363 K (90° C.). In some embodiments the concentration(c) at which the primer duplexes are used is about 1 nM, 2 nM, 3 nM, 4nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM,55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM,125 nM, 150 nM, 175 nM, 200 nM, 225 nM, 250 nM, 300 nM, 350 nM, 400 nM,450 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or 1 μM. In certainembodiments, the standard free energy of this displacement reaction plusRT ln(c) is within 3.5, 3.0, 2.5, 2.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4,0.3, 0.2, or 0.1 kcal/mol of zero kcal/mol.

In some embodiments, a primer duplex may include one or more hairpinregions that connect the complement strand to the protector strand. Incertain embodiments, the hairpin region of a primer duplex can be of anylength. In some embodiments, the hairpin region is more than 30, 25, 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3nucleotides in length. In some embodiments, the sequence of the hairpinis not complementary to a sequence of the target nucleic acid molecule.

In certain embodiments, the hairpin region has a poly-mononucleotidesequence, such as a poly-adenosine sequence, poly-deoxyadenosinesequence, a poly-5′-methyluridine sequence, a poly-thymidine sequence, apoly-guanosine sequence, a poly-deoxyguanosine sequence, a poly-cytidinesequence, a poly-deoxycytidine sequence, a poly-uridine sequence or apoly-deoxyuridine sequence.

The primer duplex described herein may be one of at least twoorientations. For example, in one orientation, the toehold region islocated at the 5′ end, immediately adjacent to the complement branchmigration region (i.e., no intervening nucleotides between the tworegions), and the complement balance region is located at the 3′ end,immediately adjacent to the complement branch migration region. In thisorientation, the protector balance region is at the 5′ end of theprotector strand, immediately adjacent to the protector branch migrationregion (FIG. 1A). In another orientation, the toehold region is locatedat the 3′ end, immediately adjacent to the complement branch migrationregion, and the complement balance region is located at the 5′ end,immediately adjacent to the complement branch migration region. In thisorientation, the protector balance region is at the 3′ end of theprotector strand, immediately adjacent to the protector branch migrationregion (FIG. 1B).

Regardless of orientation, the sequence of the complement balance regionis such that such that:

|ΔG ₁ °−ΔG ₂ °−ΔG ₃ °|≦ΔG _(R)°,

where:

-   -   ΔG₁° is the standard free energy of hybridization of the        protector balance region to the complement balance region;    -   ΔG₂° is the standard free energy of hybridization of the        protector balance region to the sequence immediately adjacent in        the first direction to the target nucleic acid sequence, if any;    -   ΔG₃° is the standard free energy of hybridization of the toehold        region to the second target nucleic acid sequence; and    -   ΔG_(R)° is 3.5 kcal/mol.

In some embodiments, a primer duplex comprises a complement strandlonger than the protector, the difference in length being dependent onthe length of the toehold region of the complement strand. The lengthsof the primers are designed such that hybridization of the complement tothe target of interest has a standard free energy) (ΔG°) close to zero.Release of the protector strand (from the primer duplex) ensures thatthis hybridization reaction is entropically neutral and robust toconcentration. As a result, in some embodiments, this reaction at roomtemperature (e.g., about 25° C. or about 298 K) parallels thespecificity of hybridization achieved at near melting temperature acrossmany conditions.

As intended herein, a ΔG° (change in standard free energy) “close tozero” refers to an absolute value (amount) less than or about 1kcal/mol, less than or about 2 kcal/mol, less than or about 3 kcal/mol,or less than or about 3.5 kcal/mol. In some embodiments, the standardfree energy of a balance region or toehold region is >−1 kcal/mol to <1kcal/mol>−3 kcal/mol to <3 kcal/mol or >−3.5 kcal/mol to <3.5 kcal/mol.

The primer duplexes may be prepared at a ratio of protector strand tocomplement strand of about 2:1 to about 5:1. In some embodiments, theratio of protector strand to complement strand is about 2:1, about 3:1,about 4:1, or about 5:1. In some embodiments, the ratio of protectorstrand to complement strand is 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1,2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1,3.6:1, 3.7:1, 3.8:1, 3.9:1, 4:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1,4.7:1, 4.8:1, 4.9:1, or 5:1. The primer duplexes may also be usedtogether with excess protector strand in any of the assays or reactionsdescribed herein. The protector strand may be in about equal to or morethan 2-, 5-, 10-, 20-, 50-, 100-, or 500-fold molar excess relative tothe primer.

Hairpin Primer Duplex Systems (e.g., Single-Stranded Systems)

In certain embodiments, a primer duplex includes a single nucleic acidthat comprises a complement region or domain, a hairpin region, and aprotector region or domain. In some embodiments, the complement domainhybridizes to the protector domain, forming a primer duplex having inintervening hairpin-loop region. Like other primer systems disclosedhere, such hairpin primer systems are designed to specifically hybridizeto a target nucleic acid molecule. Herein, a “hairpin primer duplexsystem” or a “hairpin system” includes a complement balance region, acomplement branch migration region, a toehold region, a protectorbalance region, and a protector branch migration region. As describedabove, a complement balance region is complementary to a protectorbalance region; a complement branch migration region is complementary toa protector branch migration region and a target nucleic acid region;and a toe hold region is complementary to a target nucleic acid region.A protector branch migration region corresponds to a target nucleic acidregion and is complementary to a complement branch migration region.Because the hairpin primer duplex systems described herein are formed bya single nucleic acid molecule, the design of the complement balanceregion will be dependent on the temperature and concentration at whichthe primer system is to be used, as described herein. It is to beunderstood that though the sequence of the target nucleic acid moleculemay be used to describe the characteristics of the primer systems, insome embodiments, the target nucleic acid itself may or may not be acomponent of the primer system (e.g., two-stranded or single-strandedsystems).

For primer duplexes having a hairpin region, the standard free energy ofthe confinement of the hairpin region may be considered when determiningthe standard free energy for the reaction in which the protector strandis displaced from the complement strand by the target nucleic acid.Approximate values for the standard free energy of hairpin confinementfor hairpins of various sizes are provided in Table 1 (from SantaLuciaand Hicks, Annu. Rev. Biophys. Biomol. Struct., 33:414-440, (2004)).

Hairpin Size ΔG° of Hairpin Confinement 3 nt 3.5 kcal/mol 4 nt 3.5kcal/mol 5 nt 3.3 kcal/mol 6 nt 4.0 kcal/mol 7 nt 4.2 kcal/mol 8 nt 4.3kcal/mol 9 nt 4.5 kcal/mol 10 nt 4.6 kcal/mol 12 nt 5.0 kcal/mol 14 nt5.1 kcal/mol 16 nt 5.3 kcal/mol 18 nt 5.5 kcal/mol 20 nt 5.7 kcal/mol 25nt 6.1 kcal/mol 30 nt 6.3 kcal/mol

The standard free energy of the confinement of the hairpin regionshaving lengths not provided in Table 1 (e.g., a length of n) can beestimated using the following equation:

ΔG° (loop−n)=ΔG° (loop−x)+2.44RT ln(n/x)

where ΔG° (loop−n) is the unknown standard free energy of theconfinement of a hairpin region of n nucleotides in length, ΔG° (loop−x)is the known standard free energy of the confinement of a hairpin regionof n nucleotides in length (e.g., as provided in Table 1), R is theideal gas constant, and T is the temperature at which the primer duplexis to be used. Additional information on the calculation of standardfree energies of hairpin region confinement is provided in SantaLuciaand Hicks, id., which is hereby incorporated by reference in itsentirety.

The hairpin primer duplex systems described herein may be one of atleast two orientations. For example, in one orientation shown in FIG.2A, the toehold region is located at the 5′ end of the nucleic acidmolecule. The 5′ end of the complement branch migration region isimmediately adjacent to the 3′ end of the toehold region; the 5′ end ofthe complement balance region is immediately adjacent to the 3′ end ofthe complement branch migration region; the 5′ end of the hairpin regionis immediately adjacent to the 3′ end of the complement balance region;the 5′ end of the protector balance region is immediately adjacent tothe 3′ end of the hairpin region; and the 5′ end of the protector branchmigration region is immediately adjacent to the 3′ end of the protectorbalance region. In this orientation, when the nucleic acid molecule issubjected to conditions that permit annealing, the hairpin region formsa loop that extends from the complement balance region and the protectorbalance region. In another orientation shown in FIG. 2B, the toeholdregion is located at the 3′ end of the nucleic acid molecule. The 3′ endof the complement branch migration region is immediately adjacent to the5′ end of the toehold region; the 3′ end of the complement balanceregion is immediately adjacent to the 5′ end of the complement branchmigration region; the 3′ end of the hairpin region is immediatelyadjacent to the 5′ end of the complement balance region; the 3′ end ofthe protector balance region is immediately adjacent to the 5′ end ofthe hairpin region; and the 3′ end of the protector branch migrationregion is immediately adjacent to the 5′ end of the protector balanceregion.

Regardless of orientation, the complement balance region of the hairpinprimer duplex system has a sequence such that:

|ΔG ₁ °−ΔG ₂ °−ΔG ₃ °+ΔG ₄ °+RT ln(c)|≦ΔG _(R)+,

where:

-   -   ΔG₁° is the standard free energy of hybridization of the        protector balance region to the complement balance region;    -   ΔG₂° is the standard free energy of hybridization of the        protector balance region to the sequence immediately adjacent in        the first direction to the target nucleic acid sequence, if any;        and    -   ΔG₃° is the standard free energy of hybridization of the toehold        region to the second target nucleic acid sequence;    -   ΔG₄° is the standard free energy of confinement of the hairpin        region;    -   R is the ideal gas constant;    -   T is the temperature at which the primer system is to be used;    -   c is the concentration at which the primer system is to be used;        and    -   ΔG_(R)° is 3.5 kcal/mol.

Other Primer Systems

Additional primer systems are depicted in FIGS. 4-7. Each of theseprimer systems include a complement domain and two protector domains.The complement domain has a single toehold region that is flanked by twocomplement branch migration regions and two complement balance regions.Each of the protector domains include a protector branch migrationregion that has a sequence complementary to the sequence of one of thecomplement branch migration regions, and a protector balance region thathas a sequence that is complementary to the sequence of one of thecomplement balance regions. The difference between the primer systems ofFIGS. 4-7 is in the number and location of the hairpin regions, which inturn affects the design of the complement balance regions. Like theother primer systems disclosed here, these primer systems are designedto specifically hybridize to a target nucleic acid. Though the sequenceof the target nucleic acid is used to describe the characteristics of aprimer system, in some embodiments, the target nucleic acid is not acomponent of a primer system.

As depicted in FIG. 4, a primer system may have, in 3′ to 5′ order, afirst protector domain, a first hairpin region, a complement domain, asecond hairpin region and a second protector domain.

In such embodiments, the first protector domain includes a firstprotector branch migration region and a first protector balance region.The first protector branch migration region has a sequence thatcorresponds to a first target nucleic acid sequence. The first protectorbalance region is immediately 5′ to the first protector branch migrationregion and has a sequence that does not correspond to sequenceimmediately 5′ to the first target nucleic acid sequence, if any, on thetarget nucleic acid.

In this embodiment, the first hairpin region is immediately 5′ to thefirst protector balance region.

The complement domain of such target nucleic acids comprises a firstcomplement balance region, a first complement branch migration region, atoehold region, a second complement branch migration region and a secondcomplement balance region. The first complement balance region isimmediately 5′ to the first hairpin region and has a sequencecomplementary to the sequence of the first protector balance region. Thefirst complement branch migration region is immediately 5′ to the firstcomplement balance region and has a sequence complementary to a firstprotector branch migration region. The toehold region is immediately 5′to the first complement branch migration region and has a sequence thatis complementary to a second target nucleic acid sequence that isimmediately 3′ to the first target nucleic acid sequence on the targetnucleic acid. The second complement branch migration region isimmediately 5′ to the toehold region and has a sequence complementary toa third target nucleic acid sequence that is immediately 3′ to thesecond target nucleic acid sequence on the target nucleic acid. Thesecond complement balance region is immediately 5′ to the secondcomplement branch migration region and has a sequence that is notcomplementary to sequence immediately 3′ to the third target nucleicacid sequence, if any, on the target nucleic acid.

In such embodiments, the second hairpin region is immediately 5′ to thesecond complement balance region.

In this embodiment the second protector domain includes a secondprotector balance region and a second protector branch migration region.The second protector balance region is immediately 5′ to the secondhairpin region and has a sequence complementary to the second complementbalance region. The second protector branch migration region isimmediately 5′ to the second protector balance region and has a sequencecomplementary to the second complement branch migration region.

According to this embodiment, the first complement balance region andthe second complement balance region have sequences such that:

ΔG ₁ °−ΔG ₂ °+ΔG ₃ °−ΔG ₄ °−ΔG ₅ °+ΔG ₆ °+ΔG ₇ °+RT ln(c)|≦ΔG _(R)°,

where:

-   -   ΔG₁° is the standard free energy of hybridization of the first        protector balance region to the first complement balance region;    -   ΔG₂° is the standard free energy of hybridization of the first        complement balance region to the sequence immediately 5′ to the        first target nucleic acid sequence, if any;    -   ΔG₃° is the standard free energy of hybridization of the second        protector balance region to the second complement balance        region;    -   ΔG₄° is the standard free energy of hybridization of the second        complement balance region to the sequence immediately 3′ to the        third target nucleic acid sequence, if any;    -   ΔG₅° is the standard free energy of hybridization of the toehold        region to the second target nucleic acid sequence;    -   ΔG₆° is the standard free energy of confinement of the first        hairpin region;    -   ΔG₇° is the standard free energy of confinement of the second        hairpin region;    -   R is the ideal gas constant;    -   T is the temperature at which the primer system is to be used;        and    -   c is the concentration at which the primer system is to be used;        and    -   ΔG_(R)° is 3.5 kcal/mol.

As depicted in FIG. 5, in certain embodiments, a primer system may havea hairpin primer and a protector, where the hairpin primer is a nucleicacid that includes a first protector domain, a first hairpin region, acomplement domain and the protector is a nucleic acid that includes asecond protector domain.

In such embodiments, the first protector domain includes a firstprotector branch migration region and a first protector balance region.The first protector branch migration region has a sequence thatcorresponds to a first target nucleic acid sequence. The first protectorbalance region is immediately 5′ to the first protector branch migrationregion and has a sequence that does not correspond to sequenceimmediately 5′ to the first target nucleic acid sequence, if any, on thetarget nucleic acid.

In this embodiment, the hairpin region is immediately 5′ to the firstprotector balance region.

The complement domain of such target nucleic acids comprises a firstcomplement balance region, a first complement branch migration region, atoehold region, a second complement branch migration region and a secondcomplement balance region. The first complement balance region isimmediately 5′ to the hairpin region and has a sequence complementary tothe sequence of the first protector balance region. The first complementbranch migration region is immediately 5′ to the first complementbalance region and has a sequence complementary to a first protectorbranch migration region. The toehold region is immediately 5′ to thefirst complement branch migration region and has a sequence that iscomplementary to a second target nucleic acid sequence that isimmediately 3′ to the first target nucleic acid sequence on the targetnucleic acid. The second complement branch migration region isimmediately 5′ to the toehold region and has a sequence complementary toa third target nucleic acid sequence that is immediately 3′ to thesecond target nucleic acid sequence on the target nucleic acid. Thesecond complement balance region is immediately 5′ to the secondcomplement branch migration region and has a sequence that is notcomplementary to sequence immediately 3′ to the third target nucleicacid sequence, if any, on the target nucleic acid.

In this embodiment, the second protector domain includes a secondprotector balance region and a second protector branch migration region.The second protector balance region has a sequence complementary to thesecond complement balance region. The second protector branch migrationregion is immediately 5′ to the second protector balance region and hasa sequence complementary to the second complement branch migrationregion.

According to this embodiment, the first complement balance region andthe second complement balance region have sequences such that:

|ΔG ₁ °−ΔG ₂ °+ΔG ₃ °−ΔG ₄ °−ΔG ₅ °+ΔG ₆ °|≦ΔG _(R)°,

where:

-   -   ΔG₁° is the standard free energy of hybridization of the first        protector balance region to the first complement balance region;    -   ΔG₂° is the standard free energy of hybridization of the first        complement balance region to the sequence immediately 5′ to the        first target nucleic acid sequence, if any;    -   ΔG₃° is the standard free energy of hybridization of the second        protector balance region to the second complement balance        region;    -   ΔG₄° is the standard free energy of hybridization of the second        complement balance region to the sequence immediately 3′ to the        third target nucleic acid sequence, if any;    -   ΔG₅° is the standard free energy of hybridization of the toehold        region to the second target nucleic acid sequence;    -   ΔG₆° is the standard free energy of confinement of the hairpin        region; and    -   ΔG_(R)° is 3.5 kcal/mol.

As depicted in FIG. 6, in certain embodiments a primer system may have aprotector and a hairpin primer, where the protector is a nucleic acidthat includes a first protector domain and the hairpin primer is anucleic acid that includes a complement domain, hairpin region and asecond protector domain.

In such embodiments, the first protector domain includes a firstprotector branch migration region and a first protector balance region.The first protector branch migration region has a sequence thatcorresponds to a first target nucleic acid sequence. The first protectorbalance region is immediately 5′ to the first protector branch migrationregion and has a sequence that does not correspond to sequenceimmediately 5′ to the first target nucleic acid sequence, if any, on thetarget nucleic acid.

The complement domain of such target nucleic acids comprises a firstcomplement balance region, a first complement branch migration region, atoehold region, a second complement branch migration region and a secondcomplement balance region. The first complement balance region has asequence complementary to the sequence of the first protector balanceregion. The first complement branch migration region is immediately 5′to the first complement balance region and has a sequence complementaryto a first protector branch migration region. The toehold region isimmediately 5′ to the first complement branch migration region and has asequence that is complementary to a second target nucleic acid sequencethat is immediately 3′ to the first target nucleic acid sequence on thetarget nucleic acid. The second complement branch migration region isimmediately 5′ to the toehold region and has a sequence complementary toa third target nucleic acid sequence that is immediately 3′ to thesecond target nucleic acid sequence on the target nucleic acid. Thesecond complement balance region is immediately 5′ to the secondcomplement branch migration region and has a sequence that is notcomplementary to sequence immediately 3′ to the third target nucleicacid sequence, if any, on the target nucleic acid.

According to such embodiments, the hairpin region is immediately 5′ tothe second complement balance region.

In this embodiment the second protector domain includes a secondprotector balance region and a second protector branch migration region.The second protector balance region is immediately 5′ to the hairpinregion and has a sequence complementary to the second complement balanceregion. The second protector branch migration region is immediately 5′to the second protector balance region and has a sequence complementaryto the second complement branch migration region.

According to this embodiment, the first complement balance region andthe second complement balance region have sequences such that:

|ΔG ₁ °−ΔG ₂ +ΔG ₃ °−ΔG ₄ °−ΔG ₅ °+ΔG ₆ °|≦ΔG _(R)°,

where:

-   -   ΔG₁° is the standard free energy of hybridization of the first        protector balance region to the first complement balance region;    -   ΔG₂° is the standard free energy of hybridization of the first        complement balance region to the sequence immediately 5′ to the        first target nucleic acid sequence, if any;    -   ΔG₃° is the standard free energy of hybridization of the second        protector balance region to the second complement balance        region;    -   ΔG₄° is the standard free energy of hybridization of the second        complement balance region to the sequence immediately 3′ to the        third target nucleic acid sequence, if any; and    -   ΔG₅° is the standard free energy of hybridization of the toehold        region to the second target nucleic acid sequence;    -   ΔG₆° is the standard free energy of confinement of the hairpin        region; and    -   ΔG_(R)° is 3.5 kcal/mol.

As depicted in FIG. 7, in certain embodiments a primer system may have afirst protector, a complement primer and a second protector, where thefirst protector is a nucleic acid that includes a first protectordomain, the complement primer is a nucleic acid that includes acomplement domain, and the second protector is a nucleic acid thatincludes a second protector domain.

In such embodiments, the first protector domain includes a firstprotector branch migration region and a first protector balance region.The first protector branch migration region has a sequence thatcorresponds to a first target nucleic acid sequence. The first protectorbalance region is immediately 5′ to the first protector branch migrationregion and has a sequence that does not correspond to sequenceimmediately 5′ to the first target nucleic acid sequence, if any, on thetarget nucleic acid.

The complement domain of such target nucleic acids comprises a firstcomplement balance region, a first complement branch migration region, atoehold region, a second complement branch migration region and a secondcomplement balance region. The first complement balance region has asequence complementary to the sequence of the first protector balanceregion. The first complement branch migration region is immediately 5′to the first complement balance region and has a sequence complementaryto a first protector branch migration region. The toehold region isimmediately 5′ to the first complement branch migration region and has asequence that is complementary to a second target nucleic acid sequencethat is immediately 3′ to the first target nucleic acid sequence on thetarget nucleic acid. The second complement branch migration region isimmediately 5′ to the toehold region and has a sequence complementary toa third target nucleic acid sequence that is immediately 3′ to thesecond target nucleic acid sequence on the target nucleic acid. Thesecond complement balance region is immediately 5′ to the secondcomplement branch migration region and has a sequence that is notcomplementary to sequence immediately 3′ to the third target nucleicacid sequence, if any, on the target nucleic acid.

In this embodiment the second protector domain includes a secondprotector balance region and a second protector branch migration region.The second protector balance region has a sequence complementary to thesecond complement balance region. The second protector branch migrationregion is immediately 5′ to the second protector balance region and hasa sequence complementary to the second complement branch migrationregion.

According to this embodiment, the first complement balance region andthe second complement balance region have sequences such that:

|ΔG ₁ °−ΔG ₂ °+ΔG ₃ °−ΔG ₄ −ΔG ₅ °−RT ln(c)|≦ΔG _(R)°,

where:

-   -   ΔG₁° is the standard free energy of hybridization of the first        protector balance region to the first complement balance region;    -   ΔG₂° is the standard free energy of hybridization of the first        complement balance region to the sequence immediately 5′ to the        first target nucleic acid sequence, if any;    -   ΔG₃° is the standard free energy of hybridization of the second        protector balance region to the second complement balance        region;    -   ΔG₄° is the standard free energy of hybridization of the second        complement balance region to the sequence immediately 3′ to the        third target nucleic acid sequence, if any; and    -   ΔG₅° is the standard free energy of hybridization of the toehold        region to the second target nucleic acid sequence;    -   R is the ideal gas constant;    -   T is the temperature at which the primer system is to be used;    -   c is the concentration at which the primer system is to be used;        and    -   ΔG_(R)° is 3.5 kcal/mol.

Primer Duplex Systems Lacking Balance Domains

In some embodiments, primer systems may lack balance domains. Suchnucleic acids will hybridize with a target nucleic acid with fastkinetics if the target nucleic acid has a sequence complementary to thesequence of the toehold region of the primer system, but with slowkinetics if the target nucleic is mutated so that it does not contain asequence complementary to the toehold region of the primer system. Suchprimer systems are therefore useful, for example, for locatingdifference and/or mutations in nucleic acid targets using kineticdiscrimination.

As depicted in FIG. 8, in certain embodiments a primer system mayinclude a nucleic acid having, in 3′ to 5′ order, a first protectordomain, a first hairpin region, a complement domain, a second hairpinregion and a second protector domain. The first protector domain of suchprimer systems has a sequence that corresponds to a first target nucleicacid sequence. The first hairpin region is immediately 5′ of the firstprotector domain. The complement domain has a first complement branchmigration region, a toehold region and a second complement branchmigration region. The first complement branch migration region isimmediately 5′ of the first hairpin region and has a sequencecomplementary to the branch migration sequence of the first protectordomain. The toehold region is immediately 5′ of the first complementbranch migration region and has a sequence complementary to a secondtarget nucleic acid sequence that is immediately 3′ of the first targetnucleic acid sequence on the target nucleic acid molecule. The secondcomplement branch migration region is immediately 5′ of the toeholdregion and has a sequence complementary to a third target nucleic acidsequence that is immediately 3′ of the second nucleic acid sequence. Thesecond hairpin region is immediately 5′ of the second complement branchmigration region. The second protector domain has a sequence that iscomplementary to the sequence of the second complement branch migrationregion.

Primer Modifications Generally

Each primer described herein may be comprised of DNA, RNA, or analogsthereof, and/or combinations thereof. In certain embodiments, a primercomprises one or more non-natural nucleotides. The incorporation ofnon-natural nucleotides in the primers can further augment theperformance of the primer duplexes. In particular, the protector strand,while not intended to serve to initiate transcription, may happen to becomplementary to other regions of the target or other backgroundmolecules, and may spuriously initiate replication/transcription. Toprevent this, the use of a non-natural nucleotide or a dideoxynucleotide at the 3′ end of the second protector strand may serve toprevent unintended priming by that strand. Examples of non-naturalnucleotides include, but are not limited to, iso-C, iso-G, deoxyuridine(see also Krueger et al. Chem. Biol. 16:242-48 (2009), the teachingswhich relating to non-natural nucleotides are incorporated by referenceherein).

In some embodiments, for example, in a polymerase chain reaction (PCR)where a repeated primed enzymatic function is used, the extendedcomplement strand can become a target for subsequent primerhybridization. To preserve the specificity of primer hybridization forsubsequent rounds of amplification, a balance region of a primer cannotbe replicated. Introducing a non-natural nucleotide at the interfacebetween the branch migration and balance regions of the complementstrand, for example, may prevent the balance region from beingreplicated.

In certain embodiments, the primers described herein serve as startingpoints for polymerase extensions. To facilitate analysis of amplified(nucleic acid) fragments, labeled primers can also be used in PCRreactions. Labeled primers are those that are coupled (or conjugated) toa detectable moiety. Examples include fluorescent dyes, radioactivelabels, and identifiable metals, nucleic acid sequences, and proteins.When a reaction is carried out with fluorescently labeled primers,amplicons (nucleic acid products) with a fluorescent label may begenerated.

The primers described herein can be synthesized by any method known inthe art (see, e.g., Ogilvie et al. J. Amer. Chem. Soc. 99 (23):7741-7743; Reese, C. B. Tetrahedron 34(21): 3143 (1978); Efimov et al.Nucleic Acids Res. 11(23): 8369-8387 (1983); Garegg et al. TetrahedronLett. 27(34): 4051 (1986); Beaucage et al. Tetrahedron 48(12): 2223(1992); Efimov et al. Nucleosides, Nucleotides & Nucleic Acids 26 (8-9):1087-93 (2007), incorporated herein by reference).

Target Nucleic Acid Molecules

A “target” can be a single-stranded (ss) or double-stranded (ss) nucleicacid. Target nucleic acids can be, for example, DNA, RNA, or the DNAproduct of RNA subjected to reverse transcription. In some embodiments,a target may be a mixture (chimera) of DNA and RNA. In otherembodiments, a target comprises artificial nucleic acid analogs, forexample, peptide nucleic acids (Nielsen et al. Science 254(5037):1497-500 (1991)) or locked nucleic acids (Alexei et al. Tetrahedron54(14): 3607-30 (1998)). In some embodiments, a target may be naturallyoccurring (e.g., genomic DNA) or it may be synthetic (e.g., from agenomic library). As used herein, a “naturally occurring” nucleic acidsequence is a sequence that is present in nucleic acid molecules oforganisms or viruses that exist in nature in the absence of humanintervention. In some embodiments, a target is genomic DNA, messengerRNA, ribosomal RNA, micro-RNA, pre-micro-RNA, pro-micro-RNA, viral DNA,viral RNA or piwi-RNA. In certain embodiments, a target nucleic acid isa nucleic acid that naturally occurs in an organism or virus. In someembodiments, a target nucleic is the nucleic acid of a pathogenicorganism or virus. In certain embodiments the presence or absence of atarget nucleic acid in a subject is indicative that the subject has adisease or disorder or is predisposed to acquire a disease or disorder.In certain embodiments the presence or absence of a target nucleic acidin a subject is indicative that the subject will respond well or poorlyto a treatment, such as a drug, to treat a disease or disorder.

The terms “polynucleotide,” “nucleic acid” and “nucleic acid molecule”are used interchangeably. They refer to a polymeric form of nucleotidesof any length, either deoxyribonucleotides or ribonucleotides, oranalogs thereof. Polynucleotides may have any three-dimensionalstructure, and may perform any function. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. A polynucleotide may be furthermodified, such as by conjugation with a labeling component. The term“recombinant” polynucleotide means a polynucleotide of genomic, cDNA,semi-synthetic, or synthetic origin which either does not occur innature or is linked to another polynucleotide in a non-naturalarrangement. The term “isolated nucleic acid” refers to a polynucleotideof natural or synthetic origin or some combination thereof, which (1) isnot associated with the cell in which the “isolated nucleic acid” isfound in nature, and/or (2) is operably linked to a polynucleotide towhich it is not linked in nature.

A nucleic acid may also encompass single- and double-stranded DNA andRNA, as well as any and all forms of alternative nucleic acid containingmodified bases, sugars, and backbones. The term “nucleic acid” thus willbe understood to include, but not be limited to, single- ordouble-stranded DNA or RNA (and forms thereof that can be partiallysingle-stranded or partially double-stranded), cDNA, aptamers, peptidenucleic acids (“PNA”), 2′-5′ DNA (a synthetic material with a shortenedbackbone that has a base-spacing that matches the A conformation of DNA;2′-5′ DNA will not normally hybridize with DNA in the B form, but itwill hybridize readily with RNA), and locked nucleic acids (“LNA”).Nucleic acid analogues include known analogues of natural nucleotidesthat have similar or improved binding, hybridization of base-pairingproperties. “Analogous” forms of purines and pyrimidines are well knownin the art, and include, but are not limited to aziridinylcytosine,4-acetylcytosine, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N.sup.6-methyladenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid, and2,6-diaminopurine. DNA backbone analogues provided herein includephosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene(methylimino), 3′-N-carbamate, morpholino carbamate, andpeptide nucleic acids (PNAs), methylphosphonate linkages or alternatingmethylphosphonate and phosphodiester linkages (Strauss-Soukup, 1997,Biochemistry 36:8692-8698), and benzylphosphonate linkages, as discussedin U.S. Pat. No. 6,664,057; see also OLIGONUCLEOTIDES AND ANALOGUES, APRACTICAL APPROACH, edited by F. Eckstein, IRL Press at OxfordUniversity Press (1991); Antisense Strategies, Annals of the New YorkAcademy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992);Milligan, 1993, J. Med. Chem. 36:1923-1937; Antisense Research andApplications (1993, CRC Press). The nucleic acids herein can beextracted from cells or synthetically prepared according to any meansknown to those skilled in the art; for example, the nucleic acids can bechemically synthesized or transcribed or reverse transcribed from cDNAor mRNA, among other sources.

As used herein, two nucleic acids or nucleic acid regions “correspond”to one another if they are both complementary to the same nucleic acidsequence. Two nucleic acids or nucleic acid regions are “complementary”to one another if they base-pair with each other to form adouble-stranded nucleic acid molecule.

A target nucleic acids utilized herein can be any nucleic acid, forexample, human nucleic acids, bacterial nucleic acids, or viral nucleicacids. A target nucleic acid sample can be, for example, a nucleic acidsample from one or more cells, tissues, or bodily fluids. Target samplescan be derived from any source including, but not limited to,eukaryotes, plants, animals, vertebrates, fish, mammals, humans,non-humans, bacteria, microbes, viruses, biological sources, serum,plasma, blood, urine, semen, lymphatic fluid, cerebrospinal fluid,amniotic fluid, biopsies, needle aspiration biopsies, cancers, tumors,tissues, cells, cell lysates, crude cell lysates, tissue lysates, tissueculture cells, buccal swabs, mouthwashes, stool, mummified tissue,forensic sources, autopsies, archeological sources, infections,nosocomial infections, production sources, drug preparations, biologicalmolecule productions, protein preparations, lipid preparations,carbohydrate preparations, inanimate objects, air, soil, sap, metal,fossils, excavated materials, and/or other terrestrial orextra-terrestrial materials and sources. The sample may also containmixtures of material from one source or different sources. For example,nucleic acids of an infecting bacterium or virus can be amplified alongwith human nucleic acids when nucleic acids from such infected cells ortissues are amplified using the disclosed methods. Types of usefultarget samples include eukaryotic samples, plant samples, animalsamples, vertebrate samples, fish samples, mammalian samples, humansamples, non-human samples, bacterial samples, microbial samples, viralsamples, biological samples, serum samples, plasma samples, bloodsamples, urine samples, semen samples, lymphatic fluid samples,cerebrospinal fluid samples, amniotic fluid samples, biopsy samples,needle aspiration biopsy samples, cancer samples, tumor samples, tissuesamples, cell samples, cell lysate samples, crude cell lysate samples,tissue lysate samples, tissue culture cell samples, buccal swab samples,mouthwash samples, stool samples, mummified tissue samples, autopsysamples, archeological samples, infection samples, nosocomial infectionsamples, production samples, drug preparation samples, biologicalmolecule production samples, protein preparation samples, lipidpreparation samples, carbohydrate preparation samples, inanimate objectsamples, air samples, soil samples, sap samples, metal samples, fossilsamples, excavated material samples, and/or other terrestrial orextra-terrestrial samples.

In some embodiments, a target nucleic acids utilized herein compriserepetitive sequence, secondary structure, and/or a high G/C content.

In certain embodiments, a target nucleic acid molecule of interest isabout 100 to about 1,000,000 nucleotides (nt) in length. In someembodiments, the target is about 100 to about 1000, about 1000 to about10,000, about 10,000 to about 100,000, or about 100,000 to about1,000,000 nucleotides in length. In some embodiments, the target isabout 100, about 200, about 300, about 400, about 500, about 600, about700, about 800, about 900, about 1,000, about 2,000, about 3,000, about4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9000,about 10,000, about 20,000, about 30,000, about 40,000, about 50,000,about 60,000, about 70,000, about 80,000, about 90,000, about 100,000,about 200,000, about 300,000, about 400,000, about 500,000, about600,000, about 700,000, about 800,000, about 900,000, or about 1,000,000nucleotides in length. It is to be understood that the target nucleicacid may be provided in the context of a longer nucleic acid (e.g., suchas a coding sequence or gene within a chromosome or a chromosomefragment).

In certain embodiments, a target of interest is linear, while in otherembodiments, a target is circular (e.g., plasmid DNA, mitochondrial DNA,or plastid DNA).

Combined Primer-Target Systems

In some embodiments, provided herein are primer-target systems. Aprimer-target system comprises one or more nucleic acid targets, apolymerase, and one or more primers (e.g., primer duplex and/or hairpinprimer duplex). The term “primer” encompasses any one of the primers orprimer systems described herein (e.g., single-stranded primers,double-stranded primer duplexes, and hairpin primer duplexes). Incertain embodiments, the primer-target systems described herein comprisea plurality of different primers. In some embodiments, a primer-targetsystem can comprise at least two primers, which can be used to identifyand, for example amplify, a target nucleic acid molecule. A targetnucleic acid molecule may be present amongst a plurality of non-targetnucleic acid molecules, for example, as a single copy or in low copynumber. Any one of the primer-target systems described herein maycomprises conditions similar to those used in nucleic acid amplificationor sequencing reactions (e.g., similar reagents, reaction temperature,etc.).

Methods of Use

The primer systems described herein are able to discriminate a specifictarget from spurious targets either through a thermodynamic mechanism orthrough a kinetic mechanism. In distinguishing the target from spurioustargets using a thermodynamic mechanism (described below), the stranddisplacement reaction is run to completion and the target isdistinguished from the spurious targets based on differences inequilibrium binding affinity. To distinguish the target from spurioustargets using a kinetic mechanism (described below), the stranddisplacement reaction is stopped before reaching equilibrium, and thedifferential rate in reaction completion is used to distinguish thetargets from spurious targets.

Thermodynamic Separation

The general strategy for both the thermodynamic and the kineticmechanism is to use toehold exchange strand displacement reactions. Ingeneral, toehold exchange involves extending a target's complement withan additional region that is not complementary to the target, andpre-hybridizing a protector strand to this extended complement strandand a large number of bases adjacent to the extended region, but not toa single-stranded toehold region.

FIG. 9 depicts one implementation of toehold exchange. In thisimplementation, a two-stranded primer duplex system (as described aboveand depicted in FIG. 1A) is used to distinguish between a target nucleicacid and a spurious target. The complement branch migration region andthe toehold region of the complement strand have sequences thatcorrespond to a first target sequence and a second target sequence,respectively. A complement balance region was designed to be the samelength and nucleotide base distribution as the toehold region. In thisway, the standard free energy of the strand displacement reaction shownin FIG. 9A between the correct target and the protected complement isroughly ΔG°=0 kcal/mol.

The strand displacement reaction can be written as:

-   -   Target+Complement/Protector        Target/Complement+Protector ΔG° relates to the equilibrium        constant K_(eq) by the following relation:

ΔG°=RT ln(K _(eq)).

For a reaction with ΔG°=0, the equilibrium constant (K_(eq)) is 1. Theequilibrium constant also relates to the equilibrium; for this reaction,K_(eq)=[TC][P]/[T][PC]=1. For an assay where [PC] and [P] and are inexcess of [T], [TC]/[T]=1, meaning that exactly half of all targetmolecules are hybridized at equilibrium. In the example shown in FIG.9A, ΔG°=+0.1 kcal/mol, corresponding to a K_(eq)=0.85, which means that46% of the target molecules are hybridized at equilibrium.

The protector strand correspondingly changes the standard free energy ofthe strand displacement reaction with spurious targets. In the exampleshown in FIG. 9B, the spurious target differs from the correct target bya single base, which results in the strand displacement reaction withthe same two-stranded nucleic acid primer system having a ΔG° of +3.7kcal/mol, which corresponds to K_(eq)=1.9*10⁻³. At equilibrium, only0.19% of the spurious target will be hybridized to the complement. Thus,the exemplary nucleic acid primer system depicted in FIG. 8preferentially binds to its target versus a spurious target having onlya single nucleotide mismatch by more than 200-fold.

FIG. 10 is a plot of the equilibrium binding affinity as a function ofthe standard free energy of the reaction. When the standard free energyof the reaction is very negative (as in the case in a pure hybridizationreaction), both the target and the spurious targets bind very strongly,and it is difficult to distinguish between the two, leading to falsepositives. On the other hand, when the standard free energy of thereaction is very positive, the primer binds to the target very weakly,leading to false negatives. Designing a primer duplex system to have astandard free energy of near zero results in an optimal discriminationbetween targets and spurious targets, thereby minimizing false positivesand false negatives.

Kinetic Separation

Kinetic separation relies on the differential kinetics of toeholdexchange. The kinetics of the toehold exchange reaction depend on thebinding strengths of the toeholds regions. Each kcal/mol of differencein toehold binding energy can affect kinetics by a factor of 5.4 (FIG.11), so the +3.6 kcal/mol mismatch shown in FIG. 9B would yield akinetic slowdown of 434.

Unlike in thermodynamic discrimination, kinetic discrimination occursonly when the mismatch is in the toehold region. Spurious targetsdiffering from the correct target at a position complementary to thecomplement branch migration region are unlikely to yield significantlydifferent reaction kinetics. As a consequence, methods that use thekinetic mechanism of distinguishing target from spurious target areuseful in conjunction with thermodynamic separation as a means ofpinpointing the locations of target/primer mismatches.

Significantly, primer duplex systems lacking complement balance regions,such as the primer systems depicted in FIG. 8, can be used in methodsthat exploit the kinetic mechanism to pinpoint the location oftarget/primer mismatches.

Microarrays

Nucleic acid microarrays are often used for high-throughput nucleic aciddetection, but often are unable to distinguish between closely relatednucleic acid sequences. In some embodiments, the primer duplex systemsdescribed herein can be used in nucleic acid microarrays in order to,for example, improve the specificity of microarray analysis. In someinstance, microarrays assays can be performed using methods well knownin the art, with the exception that the primer duplex systems describedherein can be used in place of conventional nucleic acid primers.

For example, as depicted in FIG. 12A, in certain embodiments, a hairpinprimer duplex system from can be directly synthesized or immobilized ona microarray chip using standard techniques. In other embodiments, atwo-stranded primer duplex system can be used in a nucleic acidmicroarray. In some embodiment, hairpin structures including twophotocleavable bases at predefined positions can be synthesized as inFIG. 12A. Subsequent exposure to light cleaves the hairpin, yielding thetwo-stranded complexes functionalized to the array surface (FIG. 12B).Other methods, such as use of nicking or restriction enzymes, can alsobe used to prepare two-stranded complexes.

Nucleic Acid Synthesis Reactions, Including Amplification Reactions

Primer duplexes and systems disclosed here can be used in someembodiments to improve the specificity of a primer-based amplificationreaction, including polymerase chain reaction (PCR), strand displacementamplification, or transcription mediated amplification, by substitutinga primer duplex system described herein for the nucleic acid primers ina primer based amplification reaction known in the art.

For example, as depicted in FIG. 13, by using as PCR primers the hairpinprimer duplex systems of the type depicted in FIG. 4, it is possible toimprove the specificity of PCR for a variety of (e.g., biotechnological)applications. In this example, a target nucleic acid sequence isamplified by forming a solution comprising a primer duplex system withthe target nucleic acid and standard reagents for performing anamplification reaction and incubating the solution under conditions suchthat an amplification reaction occurs. In certain embodiments,non-natural bases are incorporated into a hairpin primer duplex primersystems in order to prevent replication of the hairpin itself.

In some embodiments, the primer duplexes described herein can be adaptedfor use in amplifying target nucleic acids that typically requireamplification by any one or more of the following PCR methods:allele-specific PCR, assembly PCR, asymmetric PCR, helicase-dependentamplification, intersequence-specific PCR (ISSR), inverse PCR,ligation-mediated PCR, methylation-specific PCR (MSP), miniprimer PCR,multiplex PCR, nested PCR, overlap-extension PCR, quantitative PCR(Q-PCR), reverse transcription PCR (RT-PCR), solid phase PCR, thermalasymmetric interlaced PCR (TAIL-PCR), or touchdown PCR. In someinstances, the primer duplexes and methods described herein may be usedor adapted for use in any one of the foregoing PCR methods or maysubstitute (used instead of) any one of the foregoing PCR methods. Abrief description of each of the foregoing PCR methods is presentedbelow.

Allele-specific PCR is a diagnostic or cloning technique based onsingle-nucleotide polymorphisms (SNPs) (single-base differences in DNA).It typically requires prior knowledge of a DNA sequence, includingdifferences between alleles.

Assembly PCR or polymerase cycling assembly (PCA) is an artificialsynthesis of long DNA sequences by performing PCR on a pool of longoligonucleotides with short overlapping segments. The oligonucleotidesalternate between sense and antisense directions, and the overlappingsegments determine the order of the PCR fragments, thereby selectivelyproducing the final long DNA product (Stemmer et al. Gene 164(1): 49-53(1995)).

Asymmetric PCR preferentially amplifies one DNA strand in adouble-stranded DNA target. It can be used in sequencing andhybridization probing where amplification of only one of the twocomplementary strands is required (Innis et al. Proc. Natl. Acad. Sci.USA 85(24): 9436-40 (1988)).

Helicase-dependent amplification is similar to traditional PCR, buttypically uses a constant temperature rather than cycling throughdenaturation and annealing/extension cycles. DNA helicase, an enzymethat unwinds DNA, is used in place of thermal denaturation (Vincent etal. EMBO Reports 5(8): 795-800 (2004)).

Intersequence-specific PCR (ISSR) is a PCR method for DNA fingerprintingthat amplifies regions between simple sequence repeats to produce aunique fingerprint of amplified fragment lengths (Zietkiewicz et al.Genomics 20(2): 176-83 (1994)).

Inverse PCR is commonly used to identify the flanking sequences aroundgenomic inserts. It involves a series of DNA digestions andself-ligation, resulting in known sequences at either end of the unknownsequence (Ochman et al. Genetics 120 (3): 621-623 (1988)).

Ligation-mediated PCR uses small DNA linkers ligated to the DNA ofinterest and multiple primers annealing to the DNA linkers; it has beenused for DNA sequencing, genome walking, and DNA footprinting (Muelleret al. Science 246(4931): 780-786 (1988)).

Methylation-specific PCR (MSP) is used to detect methylation of CpGislands in genomic DNA. DNA is first treated with sodium bisulfite,which converts unmethylated cytosine bases to uracil, which isrecognized by primers as thymine.

Miniprimer PCR uses a thermostable polymerase (S-Tbr) and is used toamplify conserved DNA sequences, such as the 16S (or eukaryotic 18S)rRNA gene (Isenbarger et al. Applied and Environmental Microbiology74(3): 840-9. (2008)).

Multiplex-PCR targets multiple genes at once, gaining additionalinformation from a single test-run that otherwise would require severaltimes the reagents and more time to perform.

Nested PCR increases the specificity of DNA amplification, by reducingbackground due to non-specific amplification of DNA. Two sets of primersare used in two successive PCRs. In the first reaction, one pair ofprimers is used to generate DNA products, which besides the intendedtarget, may still consist of non-specifically amplified DNA fragments.The product(s) are then used in a second PCR with a set of primers whosebinding sites are completely or partially different from and located 3′of each of the primers used in the first reaction.

Overlap-extension PCR or splicing by overlap extension (SOE) is agenetic engineering technique that is used to splice together two ormore DNA fragments that contain complementary sequences. It is used tojoin DNA pieces containing genes, regulatory sequences, or mutations;the technique enables creation of specific and long DNA constructs.

Quantitative PCR (Q-PCR) is used to measure the quantity of a PCRproduct (commonly in real-time). It quantitatively measures startingamounts of DNA, cDNA, or RNA. Q-PCR is commonly used to determinewhether a DNA sequence is present in a sample and the number of itscopies in the sample.

Reverse Transcription PCR (RT-PCR) is used for amplifying DNA from RNA.Reverse transcriptase reverse transcribes RNA into cDNA, which is thenamplified by PCR. RT-PCR is widely used in expression profiling, todetermine the expression of a gene or to identify the sequence of an RNAtranscript, including transcription start and termination sites. If thegenomic DNA sequence of a gene is known, RT-PCR can be used to map thelocation of exons and introns in the gene. The 5′ end of a gene(corresponding to the transcription start site) is typically identifiedby RACE-PCR (Rapid Amplification of cDNA Ends).

Solid Phase PCR encompasses multiple meanings, including polonyamplification (where PCR colonies are derived in a gel matrix, forexample), bridge PCR (primers are covalently linked to a solid-supportsurface), conventional solid phase PCR (where asymmetric PCR is appliedin the presence of solid support bearing primer with sequence matchingone of the aqueous primers) and enhanced solid phase PCR (whereconventional solid phase PCR can be improved by employing high meltingtemperature (T_(m)) and nested solid support primer with optionalapplication of a thermal ‘step’ to favor solid support priming).

Thermal asymmetric interlaced PCR (TAIL-PCR) is used for isolation of anunknown sequence flanking a known sequence. Within the known sequence,TAIL-PCR uses a nested pair of primers with differing annealingtemperatures; a degenerate primer is used to amplify in the otherdirection from the unknown sequence (Liu et al. Genomics 25 (3): 674-81.(1995)).

Touchdown PCR (step-down PCR) is a variant of PCR that aims to reducenonspecific background by gradually lowering the annealing temperatureas PCR cycling progresses. The annealing temperature at the initialcycles is usually a few degrees (3-5° C.) above the Tm of the primersused, while at the later cycles, it is a few degrees (3-5° C.) below theprimer Tm. The higher temperatures give greater specificity for primerbinding, and the lower temperatures permit more efficient amplificationfrom the specific products formed during the initial cycles.

The temperature of the reaction solutions may be sequentially cycledbetween a denaturing state, an annealing state, and an extension statefor a predetermined number of cycles. The actual times and temperaturescan be enzyme, primer, and target dependent.

For any given reaction, denaturing states can range in certainembodiments from about 75° C. to about 100° C. The annealing temperatureand time can influence the specificity and efficiency of primer bindingto a particular locus within a target nucleic acid and may be importantfor particular PCR reactions.

For any given reaction, annealing states can range in certainembodiments from about 20° C. to about 75° C. In some embodiments, theannealing state can be performed at about 20° C. to about 25° C., about25° C. to about 30° C., about 30° C. to about 35° C., or about 35° C. toabout 40° C., about 40° C. to about 45° C., about 45° C. to about 50° C.In certain embodiments, the annealing state can be performed at roomtemperature (e.g., 20° C. or 25° C.). In some embodiments, the annealingstate can be performed at a temperature of 20° C., 21° C., 22° C., 23°C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32°C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41°C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., or50° C.

Extension temperature and time may impact the allele product yield andare understood to be an inherent property of the enzyme under study. Fora given enzyme, extension states can range in certain embodiments fromabout 60° C. to about 75° C.

In any of the foregoing embodiments, any DNA or RNA polymerase (enzymethat catalyzes polymerization of nucleotides into a nucleic acid strand)may be utilized, including thermostable polymerases and reversetranscriptases (RTases). Examples include Bacillus stearothermophiluspol I, Thermus aquaticus (Taq) pol I, Pyrccoccus furiosus (Pfu),Pyrococcus woesei (Pwo), Thermus flavus (Tfl), Thermus thermophilus(Tth), Thermus litoris (Tli) and Thermotoga maritime (Tma). Theseenzymes, modified versions of these enzymes, and combination of enzymes,are commercially available from vendors including Roche, Invitrogen,Qiagen, Stratagene, and Applied Biosystems. Representative enzymesinclude PHUSION® (New England Biolabs, Ipswich, Mass.), Hot MasterTaq™(Eppendorf), PHUSION® Mpx (Finnzymes), PyroStart® (Fermentas), KOD (EMDBiosciences), Z-Taq (TAKARA), and CS3AC/LA (KlenTaq, University City,Mo.).

Salts and buffers include those familiar to those skilled in the art,including those comprising MgCl₂, and Tris-HCl and KCl, respectively.Buffers may contain additives such as surfactants, dimethyl sulfoxide(DMSO), glycerol, bovine serum albumin (BSA) and polyethylene glycol(PEG), as well as others familiar to those skilled in the art.Nucleotides are generally deoxyribonucleoside triphosphates, such asdeoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP),deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate(dTTP), and are also added to a reaction adequate amount foramplification of the target nucleic acid.

Also provided herein are methods comprising (1) hybridizing a complementstrand of a primer duplex to a target nucleic acid, thereby dissociatingthe complement strand from its protector strand, and (2) extending thecomplement strand at its 3′ end, in a target-complementary manner, inthe presence of a polymerase.

Also provided herein are methods comprising performing a nucleic acidsynthesis reaction in the presence of a target nucleic acid, apolymerase, and one or more of the primer duplexes of any one of theembodiments described herein.

A “nucleic acid synthesis reaction” refers to any reaction in which anucleic acid is synthesized. Examples include nucleic acid amplificationreactions such as polymerase chain reaction (PCR) or a variation thereof(described elsewhere herein), a transcription reaction, a reversetranscription reaction, sequencing-by-synthesis, or other primerextension reactions (see also, Lizardi et al. Nat. Genet. 19: 225-32(1998), incorporated by reference).

In some instances, a method is provided that comprises (1) synthesizinga complement strand having a target-non-specific balance region, atarget-specific branch migration region, and a target-specific toeholdregion; (2) synthesizing a protector strand having a balance regioncomplementary to the complement strand and a branch migration regioncomplementary to the complement strand; and (3) hybridizing thecomplement strand to the protector strand to form a primer duplex.

In some instances, a method is provided that comprises (1) providing acomplement strand having a target-non-specific balance region, atarget-specific branch migration region, and a target-specific toeholdregion; (2) providing a protector strand having a balance regioncomplementary to the complement strand and a branch migration regioncomplementary to the complement strand; and (3) combining the complementstrand to the protector strand to form a primer duplex.

In some instances, a method is provided that comprises (1) providing aplurality of nucleic acid molecules comprising a target nucleic acid;(2) providing at least one primer duplex having (i) a balance region,(ii) a branch migration region complementary to the target nucleic acid,and (iii) a toehold region; and (3) combining in a single reaction theplurality of target nucleic acids, at least one primer duplex, and apolymerase under conditions suitable for nucleic acid hybridization.

Also provided herein are methods of amplifying at least one targetnucleic acid of interest, comprising (1) providing a plurality ofnucleic acid molecules comprising at least one target nucleic acid, (2)providing at least one primer duplex having (i) a balance region, (ii) abranch migration region, and (iii) a toehold region; and (3) combiningin a single reaction the plurality of target nucleic acid molecules, atleast one primer duplex, and a polymerase under conditions suitable foramplification of the at least one target nucleic acid. In certainembodiments multiple unique target nucleic acids are amplified in asingle reaction or in multiple reactions, for example, in one or moremultiplexed PCR amplification reaction. In some embodiments, about 10 to100, about 100 to about 1000, about 1000 to about 10,000, or about10,000 to about 100,000 nucleic acid targets are amplified. The numberof different primer duplexes in a reaction will depend on the number ofdesired targets.

In some embodiments, provided herein are methods of discriminatingagainst spurious nucleic acid molecules having one or more nucleotidechanges relative to a target nucleic acid molecule, comprising (1)providing a plurality of nucleic acid molecules comprising at least onetarget nucleic acid, (2) providing at least one primer duplex having (i)a balance region, (ii) a branch migration region, and (iii) a toeholdregion; and (3) combining in a single reaction the plurality of targetnucleic acid molecules, at least one primer duplex, and a polymeraseunder conditions suitable for amplification of the at least one targetnucleic acid molecule.

Any one of the methods described herein may further comprise providingor combining in a single reaction one or more of the following reagents:buffer (e.g., KCl, MgCl₂, Tris-HCl), dNTPs (e.g., dATP, dCTP, dGTP, dTTPat concentrations of, e.g., about 50 to about 100 μM), polymerase (e.g.,at concentrations of about 0.5-2.0 units per 50 μl reaction), and/orwater. The concentration of each strand of a primer duplex in a singlereaction varies depending on, for example, the concentration of targetnucleic acid. In some embodiments, about 5 to about 50 pg of plasmid orviral target may be used, or about 50 ng to about 500 ng of genomictarget may be used. In such instances, the concentration each primer(the first strand and the second strand) may be, for example, about 0.05μM to about 1 μM. In particular embodiments, the concentration of eachprimer is about 1 nM to about 1 μM.

In any one of the embodiments described herein, a single reaction may besubject to cyclic temperature changes such that a dsDNA structureundergoes multiple rounds of denaturation, subsequent primer annealing,and polymerase-based extension, for example, similar to those conditionsused for standard PCR. In some embodiments, the temperature range for adenaturation step is about 90 to about 95° C. In certain embodiments, aninitial denaturation step of about 1 to about 5 minutes is requiredprior to cycling; the exact amount of time may depend on GC content ofthe nucleic acid target of interest. In certain embodiments, thedenaturation step during a cycling reaction is about 15 to about 30seconds. In some embodiments, the temperature range for an annealingstep is about 50° C. to about 60° C. In some embodiments, the annealingstep is about 20° C. to about 40° C. in particular embodiments, theannealing step is at room temperature (about 20° C. or about 25° C.). Incertain embodiments, the annealing step during a cycling reaction isabout 15 to about 30 seconds. In some embodiments, the temperature rangefor an extension step is about 70° C. to about 75° C. In certainembodiments, the extension step during a cycling reaction is about 45 toabout 60 seconds. The temperature, time of each step, and number ofcycles of a cycling reaction may depend on the length of the nucleicacid target(s) of interest as well as the polymerase being used. Longertarget may require, for example, longer extension times. One example ofcycling conditions for a 500 nucleotide target is set forth in Table 2.

TABLE 2 1 cycle 98° C. 2 minutes 25 cycles 98° C. 15 seconds 30° C., 35°C., 40° C., 45° C., 15 seconds 50° C., 55° C., or 60° C. 72° C. 45seconds 1 cycle 72° C. 5 minutes 1 cycle  4° C. indefinite

In any one of the embodiments described herein, a single reaction (e.g.,nucleic acid amplification) may proceed at room temperature (e.g., about20° C. or about 25° C.). In certain embodiments, a single reactionproceeds at room temperature for about 1 hour.

In any one of the methods described herein, the second protector strandof a primer duplex may be provided in excess of the first complementarystrand or in excess of the annealed primer duplexes. For example, insome embodiments, the second strand is provided at a concentration about1× to about 10× (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×) theconcentration the first strand, or about 1× to about 10× (e.g., 1×, 2×,3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×) the concentration of the annealedprimer duplex. In some embodiments, the first strand is provided at aconcentration of about 0.05 μM to about 1 μM, while the second strand isprovided at a concentration of about 0.10 μM to about 2 μM, or about0.15 μM to about 3 μM, about 0.2 μM to about 4 μM, or about 0.25 μM toabout 5 μM.

Any one of the methods described herein may comprise a method selectedfrom: allele-specific PCR, assembly PCR, asymmetric PCR,helicase-dependent amplification, intersequence-specific PCR (ISSR),inverse PCR, ligation-mediated PCR, methylation-specific PCR (MSP),miniprimer PCR, multiplex PCR, nested PCR, overlap-extension PCR,quantitative PCR (Q-PCR), reverse transcription PCR (RT-PCR), solidphase PCR, thermal asymmetric interlaced PCR (TAIL-PCR), and touchdownPCR.

In any one of the methods described herein, the yield of amplifiednucleic acid target may be about 30% to about 100%. In some embodiments,the yield is at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99%, or at least 100%.

In any one of the methods described herein, the amplified nucleic acidproduct may be purified. Nucleic acid purification methods arewell-known to those of skill in the art and include, phenol extraction,guanidinium isothiocyanate, alcohol precipitation, DEAE (ion exchange),size exclusion chromatography (SEC), cesium chloride, extraction fromagarose, silica, and other column-based purification methods.

In any one of the methods described herein, a purified amplified targetnucleic acid may be about 30% to about 100% pure. In some embodiments,the purity is at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99%, or 100% pure.

Imaging

The primer duplexes and systems described herein can also be used toimprove the specificity of in situ imaging assays. Nonspecificinteractions between biological RNAs and fluorophore-labeled primers arefrequently a source of background noise. Thus, as depicted in FIG. 14,the use fluorophore-labeled nucleic acid primer systems described hereinin the place of conventional primers, in some embodiments, greatlyimproves the performance of existing in situ imaging techniques.Notably, by labeling the complement strand or domain with a fluorophoreand the protector strand or domain with a quencher, the primer duplexsystem will only produce a detectable signal when it is bound to thetarget.

Single Nucleotide Polymorphism (SNP) Detection

The accurate detection of the location and identity of single nucleotidepolymorphisms (SNPs) is of great interest for both research andtherapeutic purposes. The kinetic discrimination methods describedherein are therefore useful for the convenient identification SNPs.

Kits

Provided herein are kits comprising (1) at least one complement strandhaving a balance region, a branch migration region, and a toeholdregion, and (2) at least one protector strand having a balance regionand a branch migration region.

Provided herein are kits comprising at least one primer duplexcomprising (1) at least one complement strand or region having a balanceregion, a branch migration region, and a toehold region, and (2) atleast one protector strand or region having a balance region and abranch migration region.

Any one of the kits described herein may further comprise a polymerase.Any one of the kits provided herein may further comprise one or moreagent selected from buffer (e.g., KCl, MgCl₂, Tris-HCl), dNTPs (e.g.,dATP, dCTP, dGTP, dTTP), and water. Any one of the kits provided hereinmay comprise protector strand is molar excess of the primer. Any one ofthe kits provided herein may further comprise instructions or directionsfor obtaining instructions (e.g., from a website) for using thecomponents of the kits. Any one of the kits provided herein may furthercomprise at least one reaction tube, well, chamber, or the like.

Any one of the primers or primer systems described herein may beprovided in the form of a kit or comprised within a kit.

Examples

In accordance with the invention, the above limitations of PCR,transcription, and reverse transcription can be overcome through the useof highly specific primer duplexes. The experiments described hereindemonstrate that primer duplexes can reliably discriminate againsttargets with single-base changes (FIG. 16) for both DNA and RNA targetsand primers (FIG. 17). The correct target hybridizes to the 7/5 primerswith roughly 50% yield, but even a large excess (200×) of targets with asingle-base change is insufficient to significantly hybridize. Primerduplexes were designed and tested for multiple different targets, andeach primer duplex achieved high discrimination factors versussingle-nucleotide changes (FIG. 17). Quantitatively, the mediandiscrimination in hybridization yield to a spurious target with asingle-nucleotide change is 26.

The primer duplexes were used for PCR in a proof-of-principledemonstration (FIGS. 18A and 18B). A semi-repetitive target nucleic acidwas designed, which is difficult to amplify by traditional PCR (PCRwithout the use of the instant primer duplexes). The yield of standard21 nucleotide primers and the primer duplexes were calculated. Manydifferent thermal cycling schedules were determined in order toinvestigate the range of function. Based on the length and nucleotidecontent of the primer duplexes, standard PCR condition would predictthat the annealing temperature of the primers would 55° C. Surprisingly,as an example, even under conditions most unfavorable for primer duplexannealing (35° C. and 40° C.), the fraction (50.2%) of correct-lengthproduct amplified using the primer duplexes was higher than the fraction(31.0%) of correct-length product amplified using standard primers undertheir most favorable PCR conditions (45° C.). Furthermore, in thisparticular experiment, the primer duplexes were arbitrarily designed (7nucleotide toehold region and 5 nucleotide balance region), and were notoptimized for PCR yield performance. Thus, it is likely that even higherPCR specificity can be achieved through optimization of the instantprimer duplexes.

FIG. 15 shows highly specific PCR using the primer duplexes providedherein. In FIG. 5A, the primer “PC” is comprised of a complement strand“C” and a protector strand “P”. When PC binds to the intended target atthe correct position “X”, the single-stranded protector oligonucleotide“P” is released as an inert waste product, and the primed target iselongated by the DNA polymerase. In FIG. 5B, when the primer PC binds toan unintended target or to the correct target at an incorrect position(in either case, denoted “Y”), the displacement of the protector fromthe complementary strand “C” is thermodynamically unfavorable, andkinetically quick to reverse. Consequently, off-target amplification(e.g., amplification of Y rather than X) is expected to be significantlyreduced.

FIG. 16. shows an experimental demonstration of primer hybridizationwith single nucleotide discrimination. In FIG. 16A, short synthetic DNAtarget “X” or spurious target “Y” is reacted with the primer. (Thepoly-T tail on the protector strand “P” serves to distinguish productsfrom reactants on a gel.) Shown in red boxes are the positions ofsingle-base changes for spurious target Y. FIG. 16B shows nativepolyacrylamide gel results. The primer “PC” was prepared at a 2:1 ratioof protector P to complement C, and annealed at 1 μM concentration ofPC. Either the correct or spurious targets were added to achieve finalconcentrations of 200 nM target (X or Y), 100 nM PC, and 100 nM P. Insome embodiments, a reaction may have an excess of the protector (P)primer. For example, in some embodiments, the protector strand isprovided at a concentration of about 1× to about 10× (e.g., 1×, 2×, 3×,4×, 5×, 6×, 7×, 8×, 9×, or 10×) of the complement strand. All reactionsproceeded at room temperature (25° C.) for 1 hour. As an example, thedesignation “7/4” denotes a primer that possesses 7 nucleotides ofsingle-stranded nucleotides (as a 3′ overhang) to initiate hybridizationto the target, and the protector spontaneously dissociates 4 nucleotidesto be released. FIG. 16C is a plot of hybridization yields inferred fromdata shown in FIG. 16B. Shown as plot “X” is the hybridization of theprimer to the correct target X, while the remaining “dotted” plots showthe hybridization to the spurious targets Y. The 7/4, 7/5 and 7/6primers all discriminate in their hybridization yields (χ) between thecorrect and the spurious targets. The 7/0 target does not. In FIG. 16D,the discrimination factor (Q) is a quantitative measurement of thespecificity of the primer, and is calculated as the hybridization yield(χ) of the correct target divided by the hybridization yield (χ) of thespurious target. In 16E, there is little hybridization of the 7/5 primerto a spurious target Y even when such target is present in large excess(i.e., 200-fold).

FIG. 17. shows additional experimental results and statistics on thesingle-base discrimination abilities of primer duplexes. FIG. 17A showsthat four additional targets and sets of primers were constructed andtested: two based on naturally occurring microRNA sequences, and twodesigned to intentionally possess significant secondary structure. FIG.17B shows a histogram of the discrimination factors (Q) achieved by the7/5 primers for each target. Due to limitations of the gel scanner, itwas not possible to reliably measure discrimination factors above 100,and these were all grouped as “100+.” FIG. 17C show RNA target andprimer. The target sequence is a synthetic RNA oligonucleotide withsequence identical to the human let7g microRNA. FIG. 17D shows nativePAGE results. The PC primer was prepared at a 2:1 ratio of protector Pto complement C, and annealed at 3 μM concentration. Either the corrector spurious targets were added to achieve final concentrations of 2 μM Xor Y, 1 μM PC, and 1 μM P. The correct target successfully binds to theprimer; the hybridization yield of targets with single-nucleotidemismatches is low.

FIG. 18 shows experimental results using duplex primers to improve thePCR yield of a quasi-repetitive target. FIG. 18A shows aquasi-repetitive PCR target (168 nt) that traditional PCR primersstruggle to amplify with high yield. Here, a* is the correct target forX1. The remaining sites labeled a*m1 (which is X1-m17G), a*m2 (which isX1-m9T), and a*m3 (which is X1-m11G) are not the correct targets.Similarly, b* is the correct target for X2, and b*m1 (which is X2-m3T),b*m2 (which is X2-m11C), and b*m3 (which is X2-m18T) are not the correcttargets. Thus, the outer-most binding sites are the perfect bindingsites for the primers, but there are also 3 additional single-basemismatch primer binding sites between the perfect sites. The primerduplexes bind by 7 nucleotides to the target, and the protector mustspontaneously dissociate 5 nucleotides to be released. The primer duplexwas designed so that its 3′ end cannot be extended by the polymerase.The toehold region of the complement strand was designed at the 3′ end,instead of the 5′ end as in previous designs. In FIG. 18B, primerduplexes show significantly higher yield of correct length product, ascompared to standard primers. Each lane is labeled with the primers usedas well as the temperature cycling schedule (e.g., “98-40-72” indicatesdenaturation at 98° C., annealing at 40° C., and elongation at 72° C.).The left-most lane shows the synthetic oligonucleotide reference. Thelower numbers labeled “% Correct” indicate the relative intensity ofband corresponding to the correct length product compared to theintegrated intensity of all bands in the lane. The primer duplex PCRproduct appears as 10 nucleotides longer than the reference and thestandard PCR product because of the 5 nucleotide of overhangs (toeholdregion) on each primer.

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. The invention includes embodiments in whichexactly one member of the group is present in, employed in, or otherwiserelevant to a given product or process. The invention includesembodiments in which more than one, or all of the group members arepresent in, employed in, or otherwise relevant to a given product orprocess. Furthermore, it is to be understood that the inventionencompasses all variations, combinations, and permutations in which oneor more limitations, elements, clauses, descriptive terms, etc., fromone or more of the listed claims is introduced into another claim. Forexample, any claim that is dependent on another claim can be modified toinclude one or more limitations found in any other claim that isdependent on the same base claim.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the invention, or aspects ofthe invention, is/are referred to as comprising particular elements,features, certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements and/orfeatures. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is also noted that theterm “comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or sub-rangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

As used herein, the term “about” generally may refer to any value withina range of 10% of the recited value. In some instance, however, “about”may encompasses a range of 20% of the recited value.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the methods of the invention can be excludedfrom any one or more claims, for any reason, whether or not related tothe existence of prior art. This invention is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thedrawings. The invention is capable of other embodiments and of beingpracticed or of being carried out in various ways. Also, the phraseologyand terminology used herein is for the purpose of description and shouldnot be regarded as limiting. The use of “including,” “comprising,” or“having,” “containing,” “involving,” and variations thereof herein, ismeant to encompass the items listed thereafter and equivalents thereofas well as additional items.

Each of the foregoing patents, patent applications and references ishereby incorporated by reference, particularly for the teachingreferenced herein.

REFERENCES

-   [1] Petersen, M. & Wengel, J. LNA: a versatile tool for therapeutics    and genomics. Trends Biotechnol. 21, 74-81, (2003).-   [2] Krueger, A. T. & Kool, E. T. Redesigning the Architecture of the    Base Pair: Toward Biochemical and Biological Function of New Genetic    Sets. Chem. Biol. 16, 242-248 (2009).-   [3] Lizardi, P. M. et al. Mutation detection and single-molecule    counting using isothermal rolling-circle amplification. Nat. Genet.    19, 225-232 (1998).-   [4] Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J.,    Higuchi, R., Horn, G. T., Mullis, K. B. & Erlich, H. A.    Primer-directed enzymatic amplification of DNA with a thermostable    DNA polymerase. Science 239, 487-491 (1988).-   [5] Zhang, D. Y., Chen, X. & Yin, P. Optimizing Nucleic Acid    Hybridization Specificity. submitted (2011).

1-15. (canceled)
 16. A method comprising contacting a partiallydouble-stranded primer to a sample, and detecting hybridization of theprimer to a target nucleic acid in the sample, wherein the partiallydouble-stranded primer comprises first and second nucleic acid strandsarranged into (1) one double-stranded target-non-specific region, (2)one double-stranded target-specific region, and (3) one single-strandedtarget-specific region contributed to by the first nucleic acid strand,wherein sequences of the first nucleic acid strand that contribute toregions (2) and (3) are complementary to and bind to the target nucleicacid, and wherein the double-stranded target-non-specific region has astandard free energy approximately equal to the standard free energy forthe single-stranded target-specific region bound to the target nucleicacid.
 17. The method of claim 16, wherein the partially double-strandedprimer is labeled with a detectable moiety.
 18. The method of claim 17,wherein the detectable moiety comprises a fluorophore or a radioisotope.19. The method of claim 16, wherein the target is present as a singlecopy in the sample.
 20. A method comprising hybridizing asingle-stranded target-specific region of a first strand of a partiallydouble-stranded primer to a target nucleic acid, thereby dissociatingthe first strand of the primer from a second strand of the primer, andextending the first strand at its 3′ end, in a target-complementarymanner, in the presence of a polymerase, wherein the partiallydouble-stranded primer comprises first and second nucleic acid strandsarranged into (1) one double-stranded target-non-specific region, (2)one double-stranded target-specific region, and (3) one single-strandedtarget-specific region contributed to by the first nucleic acid strand,wherein sequences of the first nucleic acid strand that contribute toregions (2) and (3) are complementary to and bind to the target nucleicacid, and wherein the double-stranded target-non-specific region has astandard free energy approximately equal to the standard free energy forthe single-stranded target-specific region bound to the target nucleicacid.
 21. A method comprising performing a nucleic acid synthesisreaction in the presence of a target nucleic acid, a polymerase, and oneor more partially double-stranded primers, wherein the one or morepartially double-stranded primers comprises first and second nucleicacid strands arranged into (1) one double-stranded target-non-specificregion, (2) one double-stranded target-specific region, and (3) onesingle-stranded target-specific region contributed to by the firstnucleic acid strand, wherein sequences of the first nucleic acid strandthat contribute to regions (2) and (3) are complementary to and bind tothe target nucleic acid, and wherein the double-strandedtarget-non-specific region has a standard free energy approximatelyequal to the standard free energy for the single-strandedtarget-specific region bound to the target nucleic acid.
 22. The methodof claim 21, wherein the nucleic acid synthesis reaction is a nucleicacid amplification reaction.
 23. The method of claim 22, wherein thenucleic acid amplification reaction is polymerase chain reaction (PCR).24. The method of claim 21, wherein the nucleic acid synthesis reactionis a transcription reaction.
 25. The method of claim 24, wherein thetranscription reaction is a reverse transcription reaction.
 26. Themethod of claim 20, wherein two partially double-stranded primers areused. 27-34. (canceled)
 35. A method of performing a multiplexed nucleicacid amplification reaction comprising amplifying multiple uniquenucleic acid molecules using the primer of any one of claim 16.